Artificial skin tissue

Abstract

The present invention provides a method of producing an artificial skin tissue that includes fibroblasts, keratinocytes, and a platelet-derived hydrogel, matured in serum- free media comprising platelet lysate, and methods of using the same.

Description

Artificial skin tissueCross reference to related application(s)

[0001] This application claims priority from Australian provisional application no. 2023902634, the entire contents of which are incorporated herein by reference.Field of the invention

[0002] The present disclosure relates to methods of preparing an artificial skin tissue, products and uses thereof.Background of the invention

[0003] Wound repair remains a clinical challenge in both acute (eg major burns) and chronic (eg diabetic foot ulcer) injuries where the process of spontaneous healing is impaired. Skin substitutes can play a significant role in wound repair. Acellular dermal substitutes (with temporary seals) have improved clinical care for wounds that have impaired healing. Establishment of a permanent protective barrier would only be achievable by an epidermal graft, which may be derived from freshly isolated keratinocytes, culture expanded keratinocytes, or a thin donor skin graft (Sierra- Sanchez et al. 2021).

[0004] A key factor that contributes to survival of a skin substitute is its ability to vascularise. A platelet-derived hydrogel that is rich in TGF-pi , PDGF, IGF, and EGF was previously developed (Rahman et al. 2021). It was shown that this hydrogel enhances vascularisation in full thickness wounds in mice, possibly mediated by signalling through physiological concentrations of VEGF, EGF, and FGF-2 growth factors. Although platelet-derived material has previously been used for the expansion of fibroblasts, it contains growth factors (such as TGF-pi) that inhibit keratinocyte proliferation (Berndt et al. 2019; Jafar et al. 2020) and induce premature keratinocyte differentiation (Bayer et al. 2017).

[0005] Accordingly, there is a need for a skin substitute that is viable, able to vascularise, promote neo-epidermisation (a balance of keratinocyte proliferation / maturation) and provides a barrier.

[0006] Reference to any prior art in the specification is not an acknowledgment or suggestion that this prior art forms part of the common general knowledge in any jurisdiction or that this prior art could reasonably be expected to be understood, regarded as relevant, and / or combined with other pieces of prior art by a skilled person in the art.Summary of the invention

[0007] In one aspect, there is provided a method for preparing an artificial skin tissue comprising:- expanding fibroblast and keratinocyte cells from a subject, wherein the fibroblast cells are expanded in serum-free proliferation media comprising platelet lysate,- preparing a platelet-derived hydrogel from platelet precipitate,- seeding the cells within or on the platelet-derived hydrogel to form the artificial skin tissue, and- cultivating the artificial skin tissue in serum-free maturation media comprising platelet lysate.

[0008] In one preferred embodiment, the serum-free maturation media comprises about 0.1% to about 0.5% PL, preferably about 0.1% PL.

[0009] The serum-free maturation media may comprise additional components including, but not limited to, transforming growth factor pi (TGF-pi) neutralising antibody, lgG1, or combinations thereof. In one embodiment, the serum-free maturation media comprises about 10 - 50 ng / mL TGF-pi neutralising antibody.

[0010] In one preferred embodiment, the serum-free proliferation media comprises about 0.5% to about 4% PL, preferably about 2% PL.

[0011] The serum-free proliferation media may comprise additional components including, but not limited to, transforming growth factor pi (TGF-pi) neutralising antibody, lgG1, or combinations thereof. In one embodiment, the serum-free proliferation media comprises about 10 - 50 ng / mL TGF-pi neutralising antibody.

[0012] In one preferred embodiment, the keratinocytes are expanded in EpiLife media.

[0013] In any embodiment, the hydrogel is prepared using platelet precipitate (PP). Preferably, the PP is at a concentration of from about 10 - 100%, preferably, the PP is at a concentration of from about 10 - 25%.

[0014] In any embodiment, the cells may originate from a skin sample. The skin sample may be autologous, isogenic, allogenic or xenogenic. Preferably, the skin sample is obtained autologously from a subject’s own healthy skin.

[0015] In any embodiment, fibroblast cells may be seeded at a concentration of about 0.15 - 0.5 x 106 / cm2, preferably about 0.2 - 0.3 x 106 / cm2, most preferably about 0.25 x 106 / cm2.

[0016] In any embodiment, keratinocyte cells may be seeded at a concentration of about 0.3 - 1 x 106 / cm2, preferably about 0.5 - 0.7 x 106 / cm2, most preferably about 0.5 x 106 / cm2.

[0017] The artificial skin tissue may be cultivated in maturation media for about 2 - 7 days, preferably about 5 - 7 days, most preferably about 5 days.

[0018] In one embodiment, there is provided a method for preparing an artificial skin tissue comprising:- expanding fibroblast and keratinocyte cells from a subject, wherein the fibroblast cells are expanded in serum-free proliferation media comprising platelet lysate,- preparing a platelet-derived hydrogel from platelet precipitate,- seeding the cells on the platelet-derived hydrogel to form the artificial skin tissue,- cultivating the artificial skin tissue in serum-free maturation media comprising platelet lysate.

[0019] Preferably the cells are seeded on the hydrogel after gelation of the hydrogel, thereby providing cells disposed on an outer surface of the hydrogel to form the artificial skin tissue. Cells may be seeded on the hydrogel at least about 2 - 24 hours after gelation of the hydrogel.

[0020] More preferably, seeding the cells on the hydrogel comprises a first step comprising seeding the fibroblast cells on the hydrogel, and a second step comprising seeding the keratinocyte cells on the hydrogel.

[0021] In a particularly preferred embodiment, fibroblast cells are seeded on the hydrogel at least 24 hours after gelation of the hydrogel. Even more preferably, keratinocyte cells are seeded on the hydrogel about 24 hours after the fibroblast cells are seeded on the hydrogel.

[0022] In another embodiment, the method comprises:- expanding fibroblast and keratinocyte cells from a subject, wherein the fibroblast cells are expanded in serum-free proliferation media comprising platelet lysate,- seeding the fibroblast cells within a platelet-derived hydrogel prepared from platelet precipitate,- seeding keratinocyte cells on the hydrogel to form the artificial skin tissue, and- cultivating the artificial skin tissue in serum-free maturation media comprising platelet lysate.

[0023] Preferably, the fibroblast cells are seeded within the hydrogel during gelation of the hydrogel, thereby providing fibroblast cells embedded within the hydrogel.

[0024] Preferably, keratinocyte cells are seeded on the hydrogel about 24 hours after the fibroblast cells are seeded within the hydrogel.

[0025] In another aspect, there is provide an artificial skin tissue prepared by a method described herein.

[0026] The artificial skin tissue may comprise:- a platelet-derived hydrogel- cultured-expanded fibroblast and keratinocyte cells from a subject wherein the cells are embedded within, or located to one side of a surface of, the hydrogel.

[0027] In another aspect there is provided a method of treating a dermatological disease or symptom comprising contacting diseased or damaged skin with an artificial skin tissue as described herein.

[0028] In another aspect there is provided use of an artificial skin tissue as described herein in the manufacture of a medicament for the treatment of a dermatological disease or symptom.

[0029] In another aspect there is provided an artificial skin tissue as described herein for use in treating a dermatological disease or symptom comprising contacting diseased or damaged skin with the artificial skin tissue.

[0030] Diseased or loss of skin may include acute burn wounds, chronic wounds (such as diabetic foot ulcer) and during reconstructive surgery.

[0031] The artificial skin tissue may treat the wound surface, promote wound closure, replace lost skin tissue, promote growth of skin tissue, improve wound healing, prevent skin infections, reduce potential scarring of the skin, or a combination thereof.

[0032] Further aspects of the present invention and further embodiments of the aspects described in the preceding paragraphs will become apparent from the following description, given by way of example and with reference to the accompanying drawings.Brief description of the drawings

[0033] Figure 1 : Platelet lysate (PL) and platelet precipitate (PP) support expansion of dermal fibroblasts ex vivo. Primary adult fibroblast proliferation in growth media supplemented with 0.5%, 1%, 2% and 4% PL and PP, compared to 4% FBS was measured by cell counting (A&C) and Alamar Blue® (B&D) on day 2, day 4 and day 6 post-seeding. Cell numbers were determined at each split. (E&F) Concentration of Growth Factors in pooled platelet lysates (PL) and platelet precipitate (PP) from 10-12 individuals was measured by ELISA. (G) Fibroblasts were kept in media supplemented with 0.5% PL, 1% PL, 2% PL, 4% PL or 4% FBS for over 90 days. Values are represented as mean ± SEM (n = 3 per group). Data was analysed using an unpaired t-test.

[0034] Figure 2: Short term expansion of fibroblasts in PL supplemented media does not induce replication-induced senescence. Senescence in fibroblastsexpanded in 2% and 4% PL, compared to 4% FBS, was quantified by measuring (A) absolute telomere length and (B) p-galactosidase accumulation in cells. Fibroblasts in 4% FBS-supplemented standard media were kept in culture for up to 16 weeks as a reference. Values are represented as mean ± SEM (*p < 0.05, n = 5 per group).

[0035] Figure 3: PL-supplemented growth media supports expression of ECM in fibroblasts. (A&B) Expression of ECM proteins including collagen 1a1 (Col1A1), collagen 3a1 (Col3A1), collagen 4a1 (Col4A1) and fbronectin-1 (FN1), and growth factors including lnterleukin-6 (IL-6), lnterleukin-8 (IL-8), fibroblast growth factor-2 (FGF- 2) and keratinocyte growth factor (KGF) were analysed in fibroblasts expanded in 2% and 4% PL, compared to 4% FBS over 3 weeks. Values are represented as mean ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001 , n = 5 per group). (C) IL-6 and (D) IL-8 cytokine secretion by fibroblasts isolated and expanded in 2% PL-, 4% PL- or 4% FBS- supplemented media was measured by ELISA after 1 , 2, and 3 weeks of culture (n = 4).

[0036] Figure 4: Resolution of fibroblast subpopulations expanded in PL- supplemented media. Freshly isolated dermal fibroblasts were expanded in 2% PL-, 4% PL- or 4% FBS-supplemented media as a control over 4 weeks. (A) The abundance of CD90 and FAP fibroblast markers were analysed by flow cytometry. The abundance of (B) PDPN and (C) TGM2 in CD90 / FAP subpopulations were measured (n = 3 per group). (D) The gating strategy to separate CD90 and FAP subpopulations. Panels d-d” represent fibroblasts in 4% FBS, 2% PL and 4% PL after 1-2 weeks of expansion, respectively. Panels d”’-d”” represent fibroblasts in 4% FBS, 2% PL and 4% PL after 3-4 weeks of expansion, respectively.

[0037] Figure 5: Platelet-derived hydrogel was prepared using platelet precipitate (PP) to provide a functional replacement of skin ECM. (A) PP was prepared as 100%, 25%, 10% or 2.5% in phosphate-buffered saline. The gelation was initiated by adding 2.4 IIJ / mL thrombin, 1 mM CaCI2, and 0.9% NaCI. (B) PG at 100%, 25% or 10% or 2.5% in PBS incubated at 37 °C for 1 h, 2 h, 4h, or 24 h gelation.

[0038] Figure 6: Representative images of preparation of neo-dermis. Proliferation of fibroblasts when seeded on the hydrogel, 2 h or 24 h post-gelation, or mixed with the hydrogel at the time of the gelation (CT = Cells on Top, CW = Cells Within). (A) Fibroblast culture in FBS-supplemented proliferation media. (B) Fibroblast culture in PL- supplemented proliferation media.

[0039] Figure 7: Preparation of neo-dermis. Proliferation of fibroblasts when seeded on the hydrogel, 2 h or 24 h post-gelation, or mixed with the hydrogel at the time of the gelation (CT = Cells on Top, CW= Cells Within). The fibroblast-bearing hydrogel was incubated with (A&B) 4% FBS or (C&D) 2% PL-supplemented growth media. Fibroblast proliferation was measured by (A&C) cell counting and (B&D) Hoechst 3342-labelled nuclear counting, on days 2, 4, and 6. Data is representative of three independent experiments. Values are represented as mean ± SEM (**p < 0.01 , n = 3).

[0040] Figure 8: Fetal bovine serum and serum-free 3D-engineered skin structure. The expression of structural proteins were measured using immunofluorescence in HSE supplemented with (A and B) 0.3% FBS or (C and D) 0.1% PL. (A and C) Collagen IV (Col IV, a basement membrane marker) and pa n-cyto keratin (panCK) immunofluorescence staining showed higher CollV deposition in the basement membrane when fibroblasts were seeded on top (CT = cells on top) of the hydrogel. The amount of cytokeratin in the epidermis was similar whether the fibroblasts were seeded on top or within the hydrogel (CW = cells within). Integrated density quantitation for CollV and panCK measured using FIJI software are presented (n = 3). (B and D) CK5 (a marker for interfollicular stem and progenitor keratinocytes) and CK10 (a differentiation marker) immunofluorescence staining confirmed CK5 expression in basal keratinocytes, when fibroblasts were seeded on top or within the hydrogel, although at a lower level, when compared to native skin. Basal keratinocytes did not express CK10, similar to native skin, regardless of whether fibroblasts were seeded on top or within the hydrogel. EPI, epidermis; DERM, dermis; and white dotted line, dermal / epidermal junction (scale bar 100 pm). Integrated density quantitation for CK5 and CK10, measured using FIJI software, are presented (n = 4, * = p < 0.05, ** = p < 0.01 , *** = p < 0.001).

[0041] Figure 9: Optimal cell density seeding in P-HSE. Fibroblasts (0.15 x 106cells / cm2to 0.5 x 106cells / cm2) and keratinocytes (0.3 x 106cells / cm2to 1 x 106cells / cm2) were seeded on 25% PP hydrogel, 24 h post-gelation in a sequential manner and 24 h apart. Human skin equivalent (HSE) maturation media was supplemented with 0.1%, 0.2%, 0.3% PL or with 0.3% FBS and allowed to mature for (A) 5 and (B) 7 days. HSE sections were cryopreserved and stained with eosin and haematoxylin (H&E).

[0042] Figure 10: P-HSE is live in PL-supplement maturation media. Combined fibroblast and keratinocyte growth in P-HSE, day 5 in 0.1%, 0.2%, 0.3% PL or 0.3% FBS-supplemented maturation media, measured using Alamar Blue® assay.

[0043] Figure 11 : Representative confocal microscopy images of engineered skin immunostaining. P-HSE (Day 5) in 0.1%, 0.2%, 0.3% PL or 0.3% FBS-supplemented maturation media were analysed for presence of stem and progenitor cells. At each of the above supplemented media, a range of fibroblasts (0.15 x 106cells / cm2to 0.5 x 106cells / cm2) and keratinocytes (0.3 x 106cells / cm2to 1 x 106cells / cm2) were tested. Cytokeratin 5 or CK5 (a marker for interfollicular stem and progenitor keratinocytes) and cytokeratin 10 or CK10 (a maturation marker) immunofluorescence staining confirmed increased CK5 expression in basal keratinocytes. Basal keratinocytes did not express CK10. Expression pattern of CK5 and CK10 were compared to Native skin (Green = CK5, Red = CK10)

[0044] Figure 12: Autologous interfollicular stem and progenitor keratinocytes persist in a mature P-HSE. Integrated density quantitation for CK5 and CK10 of P- HSE (Day 5) in 0.1 %, 0.2%, 0.3% PL or 0.3% FBS-supplemented maturation media was measured using FIJI software.

[0045] Figure 13: Levels of additional stem / progenitor keratinocyte and fibroblast markers on day-5 post keratinocyte seeding. P-HSE in 0.1 %PL or 0.3% FBS- supplemented maturation media were analysed for presence of (A) pi integrin / CD29 (a marker for interfollicular stem and progenitor keratinocytes) and (B) vimentin (a fibroblast marker). At each of the above supplemented media, a range of fibroblasts (0.15 x 106cells / cm2to 0.5 x 106cells / cm2) and keratinocytes (0.3 x 106cells / cm2to 1 x 106cells / cm2) were seeded. Integrated density quantitation for CD29 and vimentin measured using FIJI software was applied to mark the presence of interfollicular stem and progenitor keratinocytes.

[0046] Figure 14: Functional analysis of P-HSE in vitro on the day of grafting.Luciferase Yellow (LY) penetration was used as a measure of barrier formation on partially mature P-HSE. P-HSE (Day 5) in 0.1 %, 0.2%, 0.3% PL or 0.3% FBS- supplemented maturation media, that were seeded with fibroblasts (0.15 x 106cells / cm2to 0.5 x 106cells / cm2) and keratinocytes (0.3 x 106cells / cm2to 1 x 106cells / cm2) were exposed to LY for 10 min and analysed by confocal microscopy. The yellow / greenfluorescent LY penetration through the partially mature P-HSE (day 5 of maturation) was compared to lack of LY penetration on native skin. LY penetration was quantified using FIJI software.

[0047] Figure 15: Functional analysis of fetal bovine serum and serum-free 3D- engineered skin. The barrier function was detected by confocal microscopy showing reduced Lucifer yellow (LY) penetration overtime on days 1 , 3 and 5 post keratinocytes seeding, and it was compared to lack of LY penetration on native skin. Laminin-511 (LMN-511), in red, marks the basement membrane that divides dermal and epidermal compartments. LY is shown in green and the white arrows show the depth of cells from the surface that have taken up the LY dye. The figure is a representative of two independent experiments (scale bar 100 pm).

[0048] Figure 16: H8(E staining of P-HSE. Fibroblasts 0.25 x 106 / cm2seeded on the hydrogel. Representative images on days 1, 3, 5, 8, and 11 post-seeding of 0.5 x 106 / cm2keratinocytes versus native human breast skin (bottom). EPI: epidermis, DERM: dermis (scale bar 200 pm).

[0049] Figure 17: Transmission electron microscopy analysis of (A) fetal bovine serum, (B) serum-free 3D-engineered skin, and (C) Native human skin. Atop basement membrane zone (a), tightly packed basal keratinocytes (e) formed a stratum, distinct to loosely spread fibroblasts below. Corneocytes (b), basal keratinocytes (a, e) and organelle-rich fibroblasts (c) observed across skin construct cross-section were complemented by desmosomes (d) and basal membrane ultrastructures. In between, a still disorganised basement membrane built up as a thick and non-linear entity. (C) Native human skin provided an architecturally robust tissue organisation in the basement membrane zone (a). Fibroblast cytoplasm (d) was less organelle-packed. Basement membrane (b) was a thin, linear structure, desmosomes (c) linked neighbouring keratinocytes (e), while hemidesmosomes (b) adhered basal keratinocytes to the basement membrane. Red asterisk, basement membrane; K, keratinocyte; F, fibroblast; yellow arrow, desmosome; and white arrow, hemidesmosome. Scale bar values are in pm.

[0050] Figure 18: Scanning electron microscopy (SEM) images of serum-free 3D engineered skin. A cross-sectional view of the P-HSE structure on day 5 in (A-C) 0.3% FBS and (D-F) 0.1% PL maturation media., showing various size pores in neo-dermis at(A and D) lower (scale bar 50 pm), (B and E) higher (scale bar 10 pm) magnification as well as a (C and F) top section view (scale bar 10 pm) compared to that of (G) a cross sectional (scale bar 50 pm) and (H) top view of native skin tissue (scale bar 10 pm) showing (I) a hair shaft (yellow arrow) (scale bar 50 pm).

[0051] Figure 19: Serum-free 3D skin maturation is mediated, at least partly, through TGF- 1 signalling. 3D-engineered skin was cultured 0.3% FBS (Figure 19A - 19C) or 0.1% PL (Figure 19D-19F) maturation media in in the presence of either TGFpi neutralising or lgG1 control antibodies, (a-c) Haematoxylin and eosin staining on day 5 post keratinocyte seeding, in media alone, media plus isotype control antibody, and TGF-pi neutralising antibody, respectively. Stratified epidermis was detectable even in the presence of TGF-pi antibody, (d-f) Sections were analysed for the presence of Ki67 (a marker for proliferating cells) by immunofluorescence. EPI, epidermis; DERM, dermis; white dotted line, dermal / epidermal junction (scale bar 100 pm). (Figure 19C and 19F) Ki67+ (%) cells were counted in four fields of view per experiment for statistical analysis. There was significantly more proliferation in serum-free 3D skin in presence of TGF-pi neutralising antibody compared to the isotype control antibody. Graph represents two independent experiments (* = p < 0.05).

[0052] Figure 20: Wound closure using P-HSE supplemented with FBS or PL. P- HSE seeded with the optimal fibroblasts (0.25 x 106cells / cm2) and keratinocytes (0.5 x 106cells / cm2), and were matured in media supplemented with 0.1% PL or 0.3% FBS. (A) P-HSE contraction over time was measured on day 0, day 1 and day 5 post keratinocyte seeding. (B-D) Full thickness wounds on NUDE mice were grafted with P- HSE (on day 5 of maturation) or with autologous full thickness skin graft (FTSG) and epithelisation on day 14 post skin grafts were analysed. (B) The graft area was measured over two weeks. (C) Representative images of wound closure. Full thickness wounds are presented before grafting and 14 days after grafting. (D) Representative haematoxylin and eosin stain of the P-HSE on the day of grafting (day 0) and the grafts on day 14 post skin grafting. The wounds were fully covered macroscopically after 14 days. Values are represented as mean ± SEM (*p <0.05, ***p < 0.001, n = 4-5 per group).

[0053] Figure 21 : P-HSE contributed to full thickness wound closure in NUDE mice. P-HSE supplemented with 0.3% FBS or 0.1% PL before grafting, were analysedby immunofluorescence 2 weeks post grafting using antibodies against human specific (A) human involucrin, (B) Integrin alpha6 and (C) Integrin P1 / CD29.

[0054] Figure 22: P-HSE graft take rate. Collagen deposition and CD31 (a marker for vascularisation) were analysed using (A) Masson’s trichrome (MT) and (B) CD31 immunohistochemistry, respectively. The proportion of MT or CD31 stained area against the whole wound area was calculated on day 14 post grafting. Values represent mean values ± SEM in each group (*p < 0.05, **p < 0.01 , n = 4-6 per group).

[0055] Figure 23: Water retention in P-HSE grafts. Barrier function of P-HSEs were measured (pre-graft and 14 days post-graft) and compared to native mouse skin using dermal phase meter (DMP) units according to manufacturer instructions.Detailed description of the embodiments

[0056] For purposes of interpreting this specification, terms used in the singular will also include the plural and vice versa.

[0057] As used herein, except where the context requires otherwise, the term "comprise" and variations of the term, such as "comprising", "comprises" and "comprised", are not intended to exclude further additives, components, integers or steps.

[0058] The terms "treatment" or "treating" of a subject includes delaying, slowing, stabilizing, curing, healing, alleviating, relieving, altering, remedying, less worsening, ameliorating, improving, or affecting the disease or condition, the sign or symptom of the disease or condition, or the risk of (or susceptibility to) the disease or condition. The term "treating" refers to any indication of success in the treatment or amelioration of an injury, pathology or condition, including any objective or subjective parameter such as abatement; remission; lessening of the rate of worsening; lessening severity of the disease; stabilization, diminishing of signs or symptoms or making the injury, pathology or condition more tolerable to the individual; slowing in the rate of degeneration or decline; making the final point of degeneration less debilitating.

[0059] In particularly preferred embodiments, the methods of the present invention can be to prevent or reduce the severity, or inhibit or minimise progression, of a sign orsymptom of a disease or condition as described herein. As such, the methods of the present invention have utility as treatments as well as prophylaxes.

[0060] As used herein, "preventing" or "prevention" is intended to refer to at least the reduction of likelihood of the risk of (or susceptibility to) acquiring a disease or disorder (i.e., causing at least one of the clinical signs or symptoms of the disease not to develop in an individual that may be exposed to or predisposed to the disease but does not yet experience or display signs or symptoms of the disease). Biological and physiological parameters for identifying such patients are provided herein and are also well known by physicians.

[0061] Herein, the term “subject” or “patient" can be used interchangeably with each other. The term “individual” or “patient” refers to an animal that is treatable by the artificial skin tissue and / or method, respectively, including but not limited to, for example, dogs, cats, horses, sheep, pigs, cows, and the like, as well as human, nonhuman primates. Unless otherwise specified, the “subject” or “patient” may include both male and female genders. Further, it also includes a subject or patient, preferably a human, suitable for receiving treatment with an artificial skin tissue and / or method of the present invention.

[0062] "About" as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, in some instances ±5%, in some instances ±1%, and in some instances ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0063] Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1 , 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0064] The inventors have engineered an artificial skin tissue using a subject’s own fibroblasts and keratinocytes that is effective in permanent wound closure and can be translated to clinical practice.

[0065] The inventors surprisingly found that by reducing the concentration of TGF-pi (and other growth factors) in a platelet-derived hydrogel, they were able to engineer an artificial skin tissue using the hydrogel as a scaffold to promote fibroblast and keratinocyte proliferation. The construct was able to differentiate into a mature artificial skin tissue. The artificial skin tissue was able to replace full thickness skin in mice.Methods of preparation

[0066] In one aspect, there is provided a method for preparing an artificial skin tissue comprising:- expanding fibroblast and keratinocyte cells from a subject, wherein the fibroblast cells are expanded in serum-free proliferation media comprising platelet lysate,- preparing a platelet-derived hydrogel from platelet precipitate,- seeding the cells within or on the platelet-derived hydrogel to form the artificial skin tissue, and- cultivating the artificial skin tissue in serum-free maturation media comprising platelet lysate.

[0067] In one embodiment, the serum-free maturation media comprises about 0.1 - 0.5% PL, including any range or value therein, including about 0.1% PL, 0.2% PL, 0.3% PL, 0.4% PL, or 0.5% PL, Preferably, the serum-free maturation media comprises about 0.1% PL. In one embodiment, the serum-free maturation media comprises about 0.1 - 0.3% PL, including any range or value therein, including about 0.10% PL, 0.11% PL, 0.12% PL, 0.13% PL, 0.14% PL, 0.15% PL, 0.16% PL, 0.17% PL, 0.18% PL, 0.19% PL, 0.20% PL, 0.21% PL, 0.22% PL, 0.23% PL, 0.24% PL, 0.25% PL, 0.26% PL, 0.27% PL, 0.28% PL, 0.29% PL, or 0.30% PL. Preferably, the serum-free maturation media comprises about 0.1% PL.

[0068] In one embodiment, the serum-free maturation media is substantially free of TGF-pi . Herein substantially free means the serum-free maturation media comprises about 0 ng / mL to no more than about 10 ng / mL TGF-pi , including any range or value therein, including about 0.1 ng / mL, about 0.2 ng / mL, about 0.3 ng / mL, about 0.4 ng / mL, about 0.5 ng / mL, about 0.6 ng / mL, about 0.7 ng / mL, about 0.8 ng / mL, about 0.9 ng / mL, about 1 ng / mL, about 2 ng / mL, about 3 ng / mL, about 4 ng / mL, about 5 ng / mL, about 6 ng / mL, about 7 ng / mL, about 8 ng / mL, about 9 ng / mL, or about 10 ng / mL. In one preferred embodiment, the serum-free maturation media comprises about 0.1 ng / mL to about 6 ng / mL TGF-pi .

[0069] The serum-free maturation media may comprise about 0 ng / mL to no more than about 10 ng / mL TGF-pi , including any range or value therein, including no more than about 0.1 ng / mL, no more than about 0.2 ng / mL, no more than about 0.3 ng / mL, no more than about 0.4 ng / mL, no more than about 0.5 ng / mL, no more than about 0.6 ng / mL, no more than about 0.7 ng / mL, no more than about 0.8 ng / mL, no more than about 0.9 ng / mL, no more than about 1 ng / mL, no more than about 2 ng / mL, no more than about 3 ng / mL, no more than about 4 ng / mL, no more than about 5 ng / mL, no more than about 6 ng / mL, no more than about 7 ng / mL, no more than about 8 ng / mL, no more than about 9 ng / mL, or no more than about 10 ng / mL. In one preferred embodiment, the serum-free maturation media comprises no more than about 6 ng / mL TGF-pi .

[0070] In one embodiment, the serum-free maturation media may comprise additional components including, but not limited to, transforming growth factor pi (TGF-pi) neutralising antibody, lgG1 , or combinations thereof. In one embodiment, the serum- free maturation media comprises about 10 ng / mL to about 1 pg / mL TGF-pi neutralising antibody, including any range or value therein, including about 10 ng / mL, 100 ng / mL, 200 ng / mL, 300 ng / mL, 400 ng / mL, 500 ng / mL, 600 ng / mL, 700 ng / mL, 800 ng / mL, 900 ng / mL, or 1 pg / mL. In one embodiment, the serum-free maturation media comprises at least about 10 ng / mL to about 1 pg / mL TGF-pi neutralising antibody, including any range or value therein, including at least about 10 ng / mL, 100 ng / mL, 200 ng / mL, 300 ng / mL, 400 ng / mL, 500 ng / mL, 600 ng / mL, 700 ng / mL, 800 ng / mL, 900 ng / mL, or 1 pg / mL. In a preferred embodiment, the serum-free maturation media comprises about 100 ng / mL TGF-pi neutralising antibody.

[0071] In one embodiment, the serum-free proliferation media comprises about 0.5 - 4% PL, including any value therein, including about 0.5% PL, 0.6% PL, 0.7% PL. 0.8% PL, 0.9% PL, 1.0% PL. 1.1% PL, 1.2% PL, 1.3% PL, 1.4% PL, 1.5% PL, 1.6% PL, 1.7%PL, 1.8% PL, 1.9% PL, 2.0% PL, 2.1% PL, 2.2% PL, 2.3% PL, 2.4% PL, 2.5% PL, 2.6%PL, 2.7% PL, 2.8% PL, 2.9% PL, 3.0% PL, 3.1% PL, 3.2% PL, 3.3% PL, 3.4% PL, 3.5%PL, 3.6% PL, 3.7% PL, 3.8% PL, 3.9% PL or about 4.0% PL. Preferably, the serum-free proliferation media comprises about 1 - 2% PL, preferably about 2% PL.

[0072] In one embodiment, the serum-free proliferation media is substantially free of TGF-pi . Herein substantially free means the serum-free proliferation media comprises about 0 ng / mL to no more than about 10 ng / mL TGF-pi , including any range or value therein, including about 0.1 ng / mL, about 0.2 ng / mL, about 0.3 ng / mL, about 0.4 ng / mL, about 0.5 ng / mL, about 0.6 ng / mL, about 0.7 ng / mL, about 0.8 ng / mL, about 0.9 ng / mL, about 1 ng / mL, about 2 ng / mL, about 3 ng / mL, about 4 ng / mL, about 5 ng / mL, about 6 ng / mL, about 7 ng / mL, about 8 ng / mL, about 9 ng / mL, or about 10 ng / mL. In one preferred embodiment, the serum-free proliferation media comprises about 0.1 ng / mL to about 6 ng / mL TGF-pi .

[0073] The serum-free proliferation media may comprise about 0 ng / mL to no more than about 10 ng / mL TGF-pi, including any range or value therein, including no more than about 0.1 ng / mL, no more than about 0.2 ng / mL, no more than about 0.3 ng / mL, no more than about 0.4 ng / mL, no more than about 0.5 ng / mL, no more than about 0.6 ng / mL, no more than about 0.7 ng / mL, no more than about 0.8 ng / mL, no more than about 0.9 ng / mL, no more than about 1 ng / mL, no more than about 2 ng / mL, no more than about 3 ng / mL, no more than about 4 ng / mL, no more than about 5 ng / mL, no more than about 6 ng / mL, no more than about 7 ng / mL, no more than about 8 ng / mL, no more than about 9 ng / mL, or no more than about 10 ng / mL. In one preferred embodiment, the serum-free proliferation media comprises no more than about 6 ng / mL TGF-pi .

[0074] In one embodiment, the serum-free proliferation media may comprise additional components including, but not limited to, transforming growth factor pi (TGF-pi) neutralising antibody, lgG1 , or combinations thereof. In one embodiment, the serum- free proliferation media comprises about 10 ng / mL to about 1 pg / mL TGF-pi neutralising antibody, including any range or value therein, including about 10 ng / mL,100 ng / mL, 200 ng / mL, 300 ng / mL, 400 ng / mL, 500 ng / mL, 600 ng / mL, 700 ng / mL, 800 ng / mL, 900 ng / mL, or 1 pg / mL. In one embodiment, the serum-free proliferation media comprises at least about 10 ng / mL to about 1 pg / mL TGF-pi neutralising antibody, including any range or value therein, including at least about 10 ng / mL, 100 ng / mL, 200 ng / mL, 300 ng / mL, 400 ng / mL, 500 ng / mL, 600 ng / mL, 700 ng / mL, 800 ng / mL, 900 ng / mL, or 1 pg / mL. In a preferred embodiment, the serum-free proliferation media comprises about 100 ng / mL TGF-pi neutralising antibody.

[0075] The fibroblasts may be expanded for about 5 days to about 6 weeks, including any range or value therein, for example, about 5 days, 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, about 21 days, about 4 weeks, about 5 weeks, about 6 weeks, more preferably about 4 weeks.

[0076] The fibroblasts may be expanded for about 5 days to no more than about 6 weeks, including any range or value therein, for example, no more than about 5 days, no more than 6 days, no more than about 7 days, no more than about 8 days, no more than about 9 days, no more than about 10 days, no more than about 11 days, no more than about 12 days, no more than about 13 days, no more than about 14 days, no more than about 15 days, no more than about 16 days, no more than about 17 days, no more than about 18 days, no more than about 19 days, no more than about 20 days, no more than about 21 days, no more than about 4 weeks, no more than about 5 weeks, no more than about 6 weeks, more preferably about no more than 4 weeks.

[0077] In one preferred embodiment, the keratinocytes are expanded in EpiLife media.

[0078] In any embodiment, the hydrogel is prepared using platelet precipitate (PP). Preferably, the PP is at a concentration of from about 10 - 100%, including any range or value therein, for example about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% and about 100%. Preferably, the PP is at a concentration of from about 10 - 25%. Gelation times may range from about 1 - 24 hours, including any range or value therein, for example about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, and about 24 hours.

[0079] In any embodiment, the hydrogel may be substantially free of TGF-pi (and other growth factors). In one embodiment, the hydrogel comprises about 1 to about 100 ng / mL TGF-pi , including any range or value therein, including about 1 ng / mL, 10 ng / mL, 20 ng / mL, 30 ng / mL, 40 ng / mL, or 50 ng / mL, 60ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL or 100 ng / mL. In one embodiment, the hydrogel comprises no more than about 1 to about 100 ng / mL TGF-pi , including any range or value therein, including nor more than about 1 ng / mL, 10 ng / mL, 20 ng / mL, 30 ng / mL, 40 ng / mL, 50 ng / mL, 60ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL or 100 ng / mL. In a preferred embodiment, the hydrogel comprises about 7.6 to about 76 ng / mL TGF-pi . In one embodiment, the hydrogel comprises no more than about 7.6 to about 76 ng / mL.

[0080] In one embodiment, the hydrogel may release about 0.1 to about 5 ng / mL TGF- P1 per day, including any range or value therein, including about 0.1 ng / mL, 0.2 ng / mL, 0.3 ng / mL, 0.4 ng / mL, 0.5 ng / mL, 0.6 ng / mL, 0.7 ng / mL, 0.8 ng / mL, 0.9 ng / mL, 1 ng / mL, 2 ng / mL, 3 ng / mL, 4 ng / mL or 5 ng / mL. In a preferred embodiment, the hydrogel releases about 0.5 ng / mL.

[0081] In one embodiment, the hydrogel may comprise additional components including, but not limited to, transforming growth factor pi (TGF-pi) neutralising antibody, lgG1, or combinations thereof. In one embodiment, the hydrogel comprises about 10 ng / mL to about 1 pg / mL TGF-pi neutralising antibody, including any range or value therein, including about 10 ng / mL, 100 ng / mL, 200 ng / mL, 300 ng / mL, 400 ng / mL, 500 ng / mL, 600 ng / mL, 700 ng / mL, 800 ng / mL, 900 ng / mL, or 1 pg / mL. In one embodiment, the hydrogel comprises at least about 10 ng / mL to about 1 pg / mL TGF-pi neutralising antibody, including any range or value therein, including at least about 10 ng / mL, 100 ng / mL, 200 ng / mL, 300 ng / mL, 400 ng / mL, 500 ng / mL, 600 ng / mL, 700 ng / mL, 800 ng / mL, 900 ng / mL, or 1 pg / mL. In a preferred embodiment, the hydrogel comprises about 100 ng / mL TGF-pi neutralising antibody.

[0082] In any embodiment, the hydrogel may be a substantially planar sheet. Preferably, the hydrogel has a thickness of about 2.5 - 3.5 mm, including any range or value therein, including about 2.5 mm, about 2.6 mm, about 2.7 mm, about 2.8 mm, about 2.9 mm, about 3.0 mm, about 3.1 mm, about 3.2 mm, about 3.3 mm, about 3.4 mm, or about 3.5 mm. It will be understood that the hydrogel thickness is expressed in terms of equilibrium water content and refers to the thickness of the hydrated hydrogel.Further, it will be understood that the hydrogel thickness may change, ie decrease, as the artificial skin tissue forms. The hydrogel thickness refers to the thickness of the hydrated hydrogel before the artificial skin tissue forms.

[0083] In any embodiment, the cells may originate from a skin sample. The skin sample may be autologous, isogenic, allogenic or xenogenic. Preferably, the skin sample is obtained autologously from a subject’s own healthy skin.

[0084] In any embodiment, fibroblast cells may be seeded at a concentration of about 0.15 - 0.5 x 106 / cm2, including any value or range therein, for example about 0.15 x 106 / cm2, about 0.2 x 106 / cm2, about 0.25 x 106 / cm2, about 0.3 x 106 / cm2, about 0.35 x 106 / cm2, about 0.4 x 106 / cm2, about 0.45 x 106 / cm2, about 0.5 x 106 / cm2. Preferably, fibroblast cells may be seeded at a concentration of about 0.2 - 0.3 x 106 / cm2, or about 0.5 x 106 / cm2, most preferably about 0.25 x 106 / cm2.

[0085] In any embodiment, keratinocyte cells may be seeded at a concentration of about 0.3 - 1 x 106 / cm2, including any value or range therein, for example about 0.3 x 106 / cm2, about 0.4 x 106 / cm2, about 0.45 x 106 / cm2, about 0.5 x 106 / cm2, about 0.55 x106 / cm2, about 0.6 x 106 / cm2, about 0.65 x 106 / cm2, about 0.7 x 106 / cm2, about 0.75 x106 / cm2, about 0.8 x 106 / cm2, about 0.85 x 106 / cm2, about 0.9 x 106 / cm2, about 0.95 x106 / cm2, about 1 x 106 / cm2. Preferably, keratinocyte cells may be seeded at a concentration of about 0.5 - 0.7 x 106 / cm2, or about 1 x 106 / cm2, most preferably about 0.5 x 106 / cm2.

[0086] The artificial skin tissue may be cultivated in maturation media for about 2 - 7 days, including about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days, preferably about 5 - 7 days, most preferably about 5 days.

[0087] In one embodiment, there is provided a method for preparing an artificial skin tissue comprising:- expanding fibroblast and keratinocyte cells from a subject, wherein the fibroblast cells are expanded in serum-free proliferation media comprising platelet lysate,- preparing a platelet-derived hydrogel from platelet precipitate,- seeding the cells on the platelet-derived hydrogel to form the artificial skin tissue,- cultivating the artificial skin tissue in serum-free maturation media comprising platelet lysate.

[0088] Preferably the cells are seeded on the hydrogel after gelation of the hydrogel, thereby providing cells disposed on an outer surface of the hydrogel to form the artificial skin tissue. Cells may be seeded on the hydrogel at least about 2 - 24 hours after gelation of the hydrogel, including any value or range therein, for example at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 7 hours, at least about 8 hours, at least about 9 hours, at least about 10 hours, at least about 11 hours, at least about 12 hours, at least about 13 hours, at least about 14 hours, at least about 15 hours, at least about 16 hours, at least about 17 hours, at least about 18 hours, at least about 19 hours, at least about 20 hours, at least about 21 hours, at least about 22 hours, at least about 23 hours, and at least about 24 hours after gelation of the hydrogel.

[0089] More preferably, seeding the cells on the hydrogel comprises a first step comprising seeding the fibroblast cells on the hydrogel, and a second step comprising seeding the keratinocyte cells on the hydrogel.

[0090] In a particularly preferred embodiment, fibroblast cells are seeded on the hydrogel at least 24 hours after gelation of the hydrogel. Even more preferably, keratinocyte cells are seeded on the hydrogel about 24 hours after the fibroblast cells are seeded on the hydrogel.

[0091] In another embodiment, the method comprises:- expanding fibroblast and keratinocyte cells from a subject, wherein the fibroblast cells are expanded in serum-free proliferation media comprising platelet lysate,- seeding the fibroblast cells within a platelet-derived hydrogel prepared from platelet precipitate,- seeding the keratinocyte cells on the hydrogel to form the artificial skin tissue, and- cultivating the artificial skin tissue in serum-free maturation media comprising platelet lysate.

[0092] Preferably, the fibroblast cells are seeded within the hydrogel during gelation of the hydrogel, thereby providing fibroblast cells embedded within the hydrogel.

[0093] Preferably, keratinocyte cells are seeded on the hydrogel about 24 hours after the fibroblast cells are seeded within the hydrogel.

[0094] Fibroblast and artificial skin tissue proliferation can be measured by any assay described herein.Artificial skin tissue

[0095] In one aspect, there is provide an artificial skin tissue prepared by a method described herein.

[0096] The artificial skin tissue may comprise:- a platelet-derived hydrogel- cultured-expanded fibroblast and keratinocyte cells from a subject wherein the cells are embedded within, or located to one side of a surface of, the hydrogel.

[0097] In one embodiment, the cells are located to one side of a surface of the hydrogel. Preferably the cells are disposed on an outer surface of the hydrogel. Preferably, the cells have been seeded with a preformed hydrogel to provide cells disposed on an outer surface of the hydrogel.

[0098] In an alternative embodiment, the cells are embedded within the hydrogel. Preferably, the cells have been seeded during preparation of the hydrogel to provide cells embedded within the hydrogel.

[0099] Preferably, the hydrogel component of the artificial skin tissue has a thickness of about 0.2 mm to about 1.0 mm, including any range or value therein, including about0.2 mm, about 0.3 mm, about 0.4 mm, about 0.5 mm, about 0.6 mm, about 0.7 mm, about 0.8 mm, about 0.9 mm, or about 1.0 mm. Preferably, the hydrogel component of the artificial skin tissue has a thickness of about 0.4 mm to about 0.7 mm. It will be understood that the hydrogel component of the artificial skin tissue may have a reduced thickness compared to the hydrogel used to prepare the artificial skin tissue. In other words, the hydrogel used to prepare the artificial skin tissue may have a thickness of about 2.5 - 3.5 mm. The thickness of the hydrogel may decrease as the artificial skin tissue forms such that the thickness of the hydrogel component of the artificial skin tissue may be about 0.1 to about 1.0 mm.

[0100] The artificial skin tissue may comprise an epidermis having a thickness of about 8 to 12 pm, including any range or value therein, including but not limited to, about 8 pm, about 8.5 pm, about 9 pm, about 9.5 pm, about 10 pm, about 10.5 pm, about 11 pm, about 11.5 pm, and about 12 pm. In a particularly preferred embodiment the artificial skin tissue may comprise an epidermis having a thickness of about 9.65 ± 0.5 pm.

[0101] The artificial skin tissue may form a barrier with properties comparable to native skin. The artificial skin tissue may substantially inhibit penetration of substances from one side or surface of the tissue to an opposite side or surface of the tissue. The ability of the artificial skin tissue to inhibit penetration of substances may be determined using a barrier function test, for example a luciferase yellow (LY) assay as described here. The artificial skin tissue may inhibit penetration of LY by about 70% to about 95%, including any range or value therein, including but not limited to about 70%, 75%, 80%, 85%, 90%, or 95%. The artificial skin tissue may inhibit penetration of LY by at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95%. Preferably, the artificial skin tissue may inhibit penetration of LY by about 85% or at least about 85%.Methods of treatment

[0102] In another aspect there is provided a method of treating a dermatological disease or symptom comprising contacting diseased or damaged skin with an artificial skin tissue as described herein.

[0103] Diseased or loss of skin may include a skin wound, infection. Examples of skin wounds include, burn, surgical wound, cut or scrape, areas of excised skin such as diseased skin, chemical burn, infected skin or tissue, ulcer, or wounds treated or created by reconstructive surgery.

[0104] The artificial skin tissue may treat the wound surface, promote wound closure, replace lost skin tissue, promote growth of skin tissue, improve wound healing, prevent skin infections, reduce potential scarring of the skin, or a combination thereof.

[0105] In a particularly preferred embodiment, there is provided a method of promoting wound closure in a subject comprising contacting the wound with an artificial skin tissue described herein thereby grafting the wound with the tissue.

[0106] The artificial skin tissue may be integrated with the subject’s skin after grafting.

[0107] The artificial skin tissue constructs when containing autologous keratinocytes, can be grafted and close a wound definitively. They can also be used as biological wound dressings with either allogenic or autologous keratinocytes in the construct.

[0108] In one embodiment, the method increases the amount of skin growth or repair factor comprising any one or more keratinocyte growth factor (KGF), IL-6, I L-1 a, IL-1 b, periostin, Ki67, involucrin, loricrin, p63, pi integrin, keratin 5, keratin 14, or connexion 43.

[0109] The artificial skin tissue constructs may be used for subjects of all ages, such as adults, young adults, or for paediatric use. In one embodiment, the artificial skin construct is for use in adults or young adults. In another embodiment, the artificial skin construct is for paediatric use.

[0110] It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention.ExamplesMethodsIsolation and expansion of adult keratinocytes and dermal fibroblasts

[0111] Keratinocytes were isolated and cultured as previously described (Banakh et al. 2020). Dermal fibroblasts were isolated and cultured as described by Boyce (1999) with minor modifications. Isolated cells were seeded and expanded in low glucose DMEM with hydrocortisone (0.5 pg / mL, Merck, Billerica, USA), insulin (50 lU / mL, Novo Nordisk, Australia), gentamicin (50 pg / mL, Life Technologies, Carlsbad, USA) and FBS (4%, Cytiva, Marlborough, USA) or PL (0.5%, 1%, 2% or 4%, Australian Red Cross Lifeblood).IL-6 and IL-8 ELISA

[0112] Human fibroblasts were cultured in 6-well plates for 3 weeks with 4% FBS, 2% PL and 4% PL-supplemented medium. Supernatants were assessed for human IL-6 and IL-8 cytokine content using ELISA (BD Bioscience, San Diego, USA) according to the manufacturer’s instructions.Transmission electron microscopy (TEM)

[0113] Tissue samples were cut into cubes of 1 mm3and placed into primary fixative, consisting of 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 M sodium cacodylate buffer, for 1 h at room temperature followed by overnight incubation at 4 °C. The tissues were then rinsed in fresh 0.1 M sodium cacodylate buffer three times for 15 min each. Secondary fixation was performed using 1% osmium tetroxide and 1.5% potassium ferricyanide in 0.1 M cacodylate buffer for 1 h at room temperature. The tissues were rinsed in three washes of milli-Q water for 15 min each. The fixed tissues were dehydrated by incubating in increasing concentrations of ethanol for 15 min, consisting of 30, 50, 70, 90, and 100% ethanol. The ethanol was removed and replaced with 100% propylene oxide. Dehydrated tissues were incubated in a mixture of Epon resin and propylene oxide at a ratio of 1:1 for 6 h at room temperature, followed by a 2:1 Epon / propylene oxide mixture overnight. Tissues were incubated in 100% freshly made Epon resin for 6 h, followed by another 100% resin change overnight. The tissues were then placed into Beem capsules in 100% resin, and the resin was polymerised for 48 h in an oven at 60 °C. Resin-embedded tissue was sectioned with a Diatome diamond knife using a Leica UCS ultramicrotome. Sections of thickness 70-90 nm were collected onto 150 mesh copper / palladium grids and stained sequentially with 1% uranyl acetatefor 5 min and lead citrate for 5 min. The sections were imaged in a JEOL 1400 + transmission electron microscope at 80 kV, and images were captured with a digital camera at a resolution of 2 K x 2 K.Scanning electron microscopy (SEM)

[0114] All biological samples were fixed with 4% PFA frozen in OCT at - 80°C. The sections were dehydrated with ascending concentrations of ethanol (50%, 75%, 95%, 100%) for 15 min each. All samples were treated with hexamethyldisilazane (HMDS), air-dried overnight and mounted on a conductive aluminium surface using carbon tapes. Samples were coated with a thin gold layer (Bal- Tec SCD 005, Leica USA) prior to examination under the scanning electron microscope (Nova Nano- SEM, FEI, USA). Images were acquired at 5 mm working distance under a beam of 10 kV and quantified using Image J software (NIH).Platelet-rich Human Skin Equivalent (P-HSE)

[0115] Dermal fibroblasts were seeded either (i) within the hydrogel at the time of gelation or (ii) on top of the hydrogel after gelation. Keratinocytes were seeded on top of the hydrogel and 3D skin was cultivated in maturation medium supplemented with 0.3% FBS or 0.1% PL for up to 11 days (Boyce 1999). HSE maturation media was prepared based on UCDM1 media (Boyce et al, 2017) modified with a low glucose DMEM (Sigma-Aldrich, St. Louis, MO) and added hydrocortisone (0.5 pg / mL, Merck, Billerica, USA), Ham's F12 (Gibco, NY, USA), L-Glutamine (4.5 mM, Sigma-Aldrich, St. Louis, MO), L-Serine (1.5 mM, Sigma-Aldrich, St. Louis, MO), stock 11a (0.1%, Sigma-Aldrich, St. Louis, MO), CaCh (1 mM, Phebra, NSW, AU), adenine (1.8 mM, Calbiochem, San Diego, CA), strontium chloride hexahydrate (1 mM, Sigma-Aldrich, St. Louis, MO), FBS (4%, Cytiva, Marlborough, USA) or PL (0.1%, 0.2% or 0.3%), ITS (1%, Sigma-Aldrich, St. Louis, MO), Linoleic acid (10 pg / mL, Sigma-Aldrich, St. Louis, MO), Ascorbic acid-2- phosphate (0.1 mM, Sigma-Aldrich, St. Louis, MO), triiodothyronine (20 pM, Sigma- Aldrich, St. Louis, MO), Hydrocortisone (0.5 pg / mL, Calbiochem), KGF (2.5 ng / mL, R&D Biosystems, Minneapolis, MN, USA), bFGF (1 ng / mL, R&D Biosystems, Minneapolis, MN, USA ), gentamicin (50 pg / mL, Life Technologies, Carlsbad, USA) and EGF (10 ug / mL, R&D Biosystems, Minneapolis, MN, USA). Where noted, transforming growth factor pi (TGF-pi) neutralising antibody (Abeam, Waltham, USA) or lgG1 (DAKO, Glostrup, Denmark) was added to media (1 :10,000) during the cultivation period.Platelet derived lysate and hydrogel preparation

[0116] Platelet lysate (PL) batches were produced from platelet concentrates (Australian Red Cross Lifeblood) of either a single blood group or a mixed blood group. Platelet concentrates were frozen at -80 °C within one day after expiry. Platelets were thawed at 2-6 °C for 24 h, and centrifugation was used to remove debris or precipitate (5005 g, 10 min). Freezing, thawing, and centrifugation processes were repeated. Batches of PL were pooled from 10-12 individual concentrates. The pooled lysate was then frozen at -80 °C. Platelet-derived hydrogel was prepared from platelet precipitate (PP) as previously described (Rahman et al, 2021). Concentrations of growth factors were measured using Quantikine ELISA kits (R&D Systems, Minneapolis, MN, USA) in accordance with manufacturer’s instructions.Proliferation assay

[0117] Dermal fibroblasts and P-HSE proliferation was measured using Alamar Blue® (Bio-Rad Laboratories Inc, Hercules, USA) in accordance with manufacturer’s instructions. Fibroblasts in a monolayer and P-HSE were cultured in triplicate in a 24- well plate. The culture media was removed and replaced with 10% Alamar Blue® stock in media, and the cells were incubated at 37 °C for 2 h. 100 pL aliquots from each well were transferred to a 96-well plate. 590 nm fluorescence emission was measured using a Fluostar Optima plate reader (BMG Labtech, Ortenberg, Germany). Long-term fibroblast proliferation was determined by cell counting and trypan blue dye exclusion, wherein 70-80% confluence was reached at each split.Senescence

[0118] Senescence-associated p-galactosidase deposits were detected as described (Dimri et al, 1995). Briefly, 30 x 103fibroblasts were seeded on glass coverslips. Cells were fixed in 3% formaldehyde (3 min) and incubated in fresh p-Galactosidase staining solution at 37 °C, overnight. Stained coverslips were washed in PBS and observed under a light microscope to identify p-galactosidase-positive cells.Absolute telomere length measurement

[0119] Dermal fibroblast’s DNA was extracted using QIAmp (Qiagen, Hilden, Germany) kit and quantitatively measured with a Quantus Fluorometer using a QuantiFluor ONEdsDNA kit. Quantitative PCRs were performed as previously described (O’Callaghan and Fenech, 2011). Briefly, the amount of DNA was measured in triplicate in 96-well plates, with 20 ng DNA per well added to GoTaq qPCR Master mix (Promega), and 5'cggtttgtttgggtttgggtttgggtttgggtttgggtt-3' forward and 5'ggcttgccttacccttacccttacccttacccttaccct-3' reverse primers. Standard curves were generated from the dilution of known quantities of telomere standard, 5'-(TTAGGG )14- 3'. pBR322 plasmid (Life Technologies) was used to standardise DNA content to 20 ng for consistency.Growth factor and ECM expression

[0120] RNA was extracted using Trizol as previously described (Banakh et al. 2020). cDNA was prepared from 400 ng of RNA using a GoScript Reverse Transcriptase Kit (Promega, Madison, USA). qPCR was performed in a LightCycler® 480 Multiwell Plate 384 (Roche, Basel, Switzerland) using Go Taq qPCR Master Mix (Promega, Madison, USA) according to the manufacturer’s instructions. Primers used to detect growth factors, ECM markers and housekeeping genes in fibroblasts were as follows: KGF (5- ttgtggcaatcaaaggggtg-3', 5'-cctccgttgtgtgtccatttagc-3'), FGF-2 (5- gaagagcgaccctcacatcaagcta-3', 5'-cagttcgtttcagtgccacatacc-3'), IL-6 (5'-ggt- acatcctcgacggcatct-3', 5'-gtgcctctttgctgctttcac-3'), IL-8 (5'-actgagagtgattgagagtggac-3', 5'-aaccctctgcacc- cagttttc-3'), Col1A1 (5'-atgtctagggtctagacat- gttca-3', 5'- ccttgccgttgtcgcaga-3'), Col3A1 (5'-agctgttgaaggaggatgttcc-3', 5'- ctgcgagtcctcctactgct- 3'), FN1 (5'-caactcactgacctaagctttgt-3', 5'-ggtgaatcgcaggtcagt-3'), GAPDH (5- ctctgctcctcctgttcgac-3', 5'-aaatgagccccagccttctc-3'), HPRT (5 - attggtaatgaccagtaccagtcaacag-3', 5'-gcattgttttgccagt- gtcaa-3') and TBP (5- cacgaaccacggcactgatt-3', 5'-ttttcttgctgc- cagtctggac-3').Flow cytometry

[0121] Dermal fibroblasts cultured in 4% FCS, 2% PL or 4% PL supplemented media were mixed with Efluor 450 viability dye (Thermo Fisher, Eugene, USA) for 30 min at 4°C. Cell aliquots were washed in PBS after fixation, label- ling and permeabilization steps. Cells were fixed in 1% formaldehyde and blocked in 2% bovine serum albumin (MP Biomedicals, Auckland, New Zealand) / 2% FBS, 15 min at 4°C. Cells were stained with CD90-BV605 antibody (BioLegend, San Diego, USA, 1:10) and FAP- APC (R&D Systems, Minneapolis, USA, 1 :10) for 30 min at 4°C under agitation. Samples werepermeabilised in 0.2% Triton-X (Sigma-Aldrich). Cells were then incubated with mouse anti-vimentin (DAKO, 1 :100), rabbit anti-podoplanin (Abeam, Cambridge, USA, 1:50) and mouse anti-transglutaminase 2 (Abeam, 1 :100) antibodies, followed by incubation with anti-mouse Alexa Fluor 488 (Invitrogen, Waltham, USA, 1 :400) and anti-rabbit Ig- PE (Invitrogen, 1:400). Analysis was carried out using Fortessa (BD Biosciences) and Flowlogic software (Inivai Technologies, Mentone, Australia).Histological analysis

[0122] P-HSE biopsies were cut in the midline, sectioned on days 5, and 7 and stained with haematoxylin and eosin and Masson’s trichrome stain according to standard protocols. Slides were imaged using an Olympus BX51 microscope and Olympus DP70 colour camera. Fresh collagen deposition in neo-dermis was quantified by Masson’s trichrome stain at the wound boundaries and the middle of the wound using Image J (NIH).Immunofluorescence (IF)

[0123] Cryopreserved sections were blocked with 5% BSA with 5% donkey serum (Sigma-Aldrich, St. Louis, MO) and incubated overnight with primary antibodies: rabbit anti-human K5 (1:500, Biolegend), mouse anti-human K10 (1 :500, DAKO), rabbit antihuman CD29 (1:200, GeneTex, Irvine, USA), or mouse anti-human Vimentin (1:200, DAKO). Slides were washed and incubated with Alexa Fluor-conjugated mouse or rabbit secondary antibodies (1 :400 BD Biosciences, Columbus, USA). Sections were imaged on a Nikon Ti-E microscope. Staining was quantitatively measured to obtain the integrated density using FIJI software (NIH). Thresholds were set using the negative control stain and adjusted to minimise background.Lucifer yellow barrier function

[0124] Fresh P-HSEs were incubated with Lucifer yellow (1 mg / mL, Sigma-Aldrich, St. Louis, MO) for 10 min at room temperature on days 1 , 3, and 5. Excess dye was blotted and tissues were fixed in paraformaldehyde (PFA). Sections were stained for laminin- 511 and imaged by confocal microscopy. Penetration of fluorescent dye through the P- HSE surface was quantified using image J.Murine Surgery

[0125] The protocol and procedures were ethically reviewed and approved by the Alfred Research Alliance Animal Ethics Committee (AEC E / 1920 / 2019 / A), and was in accordance with the NIH Guide for Care and Use of Laboratory Animals. Generally, the procedure was performed as previously described, with some modifications (Dunn et al, 2013). Briefly, 10-12 week-old NUDE mice were anaesthetised with isoflurane (2 L / min) and a full-thickness surgical wound created by excising a circular section of skin 0.8 mm in diameter on the dorsal side, 1 cm on either side of the midline. A custom-made silicon (Life Technologies, Carlsbad, CA, USA) ring was adhered to the outer edge of the wound using Histoacryl skin glue (B. Braun Surgical, Barcelona, Spain) and sutured (monofilament nylon, B. Braun Surgical) with 6-8 simple interrupted stitches along the outer circumference of the ring. The P-HSE was applied to the wound and left without disturbance for a maximum five days. The wound was dressed and sealed with SurfaSoft® (Tauren, Rijswijk, Netherlands), Tegaderm™ (3 M Health Care, St. Paul, MN, USA), and Nexcare™ (3 M Health Care) for the duration of the experiment. Mice were sacrificed and the grafts were analysed after two weeks.Results

[0126] The growth of fibroblasts over the short and long-term is represented in Figure 1. Fibroblasts grow in proliferation media supplemented with PL or with PP (range 0.5%-4%) at a comparable rate to FBS (4%). However, at a lower concentration (0.5%) they grow more slowly than when cultured in FBS-supplemented media over the short- (6 days) and long-term (90 days). This difference was not significant. Levels of growth factors in platelet lysate and platelet precipitate are represented in Fig. 1E and 1 F. The most abundant growth factors were the a-granule factors, including TGF-pi, platelet- derived growth factor 1 (PDGF) and insulin-like growth factor 1 (IGF-1).

[0127] The rate of fibroblast growth in PL- (2% and 4%) supplemented media was similar to that in FBS-supplemented media in the short-term (Fig. 1A and 1 B). In the long-term (Fig. 1G), the growth rate of fibroblasts declined in lower concentrations of PL-supplemented media, which led to a drop below the number required for cell splitting after 21 (0.5%), 49 (1% and 2%), and 63 (4%) days. Fibroblasts cultured in 4% FBS continued to grow over 90 days. There were no obvious morphological differences between dermal fibroblasts cultured in FBS and 2% PL. However, long term culture in 4% PL caused stress, which was evident from increased cell membrane filopodia.Subsequently, 2% PL was used to supplement media for short-term, serum-free fibroblast expansion.

[0128] Replication-induced senescence in long-term expanded fibroblasts was measured using two independent methods. Firstly, absolute telomere length of fibroblasts cultured in 2% or 4% PL-supplemented media and FBS-supplemented media was determined by qPCR (Fig. 2A). As expected for ex vivo cell culture, absolute telomere length declined over 8 weeks regardless of supplementation. The rate of decline for 2 or 4% PL supplementation was comparable to 4% FBS for up to 6 weeks. Secondly, p-galactosidase was measured using p-gal staining (Fig. 2B). There was an increase in the number of p-gal-positive fibroblasts after culturing in 2% PL for 7-8 weeks (p = 0.0201). No increase was observed at earlier time points. Overall, these data suggest that replication-induced senescence may be present when fibroblasts are cultured in PL over a longer period of time. However, there is no evidence that culturing fibroblasts in PL-supplemented media for a short period of time (up to 6 weeks) triggers senescence.

[0129] ECM protein and growth factor expression in PL-expanded fibroblasts was measured (Fig. 3A and 3B). After 3 weeks of culture, the expression levels of Col III, Col IV, and fibronectin in fibroblasts expanded in PL-supplemented media was comparable to that of fibroblasts expanded in FBS-supplemented media. PL induced higher Col I expression in fibroblasts compared to FBS, after 3 weeks (p < 0.0001). PL did not cause any increase of IL-6 and IL-8 cytokines in fibroblasts compared to FBS at the protein level, suggesting that inflammation was not induced (Fig. 3B-3D). Expression of FGF-2 and keratinocyte growth factor (KGF) supports ex vivo growth of keratinocytes, and was measured by qPCR in fibroblasts cultured in PL-supplemented media (Fig. 3B). Expression of FGF-2 and KGF remained unchanged.

[0130] Fluorescence-activated cell sorting (FACS) was employed to monitor phenotypic changes in PL-expanded fibroblasts. Cells were resolved into four subpopulations based on their CD90 and FAP surface marker expressions (Fig. 4D). The proportion of CD90+FAP“ (reticular phenotype), CD90+FAP+(intermediate phenotype) and CD90“FAP+(papillary phenotype) fibroblasts were determined after 1-2 weeks and 3-4 weeks of growth in PL vs FBS. Fig. 4A shows a small proportion of CD90+FAP+fibroblasts (6.8% ± 0.1%) after 1-2 weeks in FBS. This subpopulationincreased to 65.5% (± 0.2%) after the fibroblasts were cultured for 1-2 weeks in PL and remained high (73.8% ± 0.1%) when cultured in PL up to 3-4 weeks. Conversely, the majority of fibroblasts in FBS for 1-2 weeks were CD90+FAP“ (80.1% ± 0.1%), but their abundance decreased to 32.5% (± 0.1%) after 3-4 weeks in culture. A similar decrease in the CD90+FAP“ subpopulation (23.2% ± 0.2%) was observed when FBS was substituted with PL for 1-2 weeks and remained low (19.5 ± 0.1%) after 3-4 weeks. CD90“FAP+papillary fibroblasts accounted for < 1% of the total population in FBS and PL cultures. Therefore, they were not analysed further due to low abundance.

[0131] In order to further characterise the PL-expanded fibroblasts, the expression of podoplanin (PDPN) and transglutaminase (TGM2) was determined in CD90+FAP+and CD90+FAP“ subpopulations (Fig. 4B and 4C). PDPN, a mucin-type transmembrane protein expressed in dermal fibroblasts, was expressed in the majority of CD90+FAP+(intermediate phenotype) fibroblasts (95.7% ± 0.1%) after 1-2 weeks expansion in FBS. Its expression remained high in CD90+FAP+subpopulation, when fibroblasts were expanded in 2% PL (94.6% ± 0.1%) and 4% PL (98.3% ± 0.1%) for 1-2 weeks. There was lower PDPN in CD90+FAP“ reticular fibroblasts expanded in FBS (64.7% ± 0.1%). However, after 1-2 weeks in 2% PL and 4% PL, CD90+FAP“ fibroblasts showed an increase in PDPN to 78.1% (± 0.1%) and 89.7% (0.1%), respectively. Longer 3-4-week expansion of CD90+FAP“ reticular fibroblasts in 2% PL did not change PDPN level (72.8% ± 0.1%), whereas 4% PL reduced PDPN levels to 52.9% (± 0.1%).

[0132] Unlike PDPN, TGM2 expression was not sustained in culture after 1-2 weeks, regardless of the media supplement. TGM2 decreased from 32.5% after 1-2 weeks to 0.1% after 3-4 weeks, in the CD90+FAP+intermediate subpopulation, when expanded in FBS. A similar TGM2 decrease was observed after 3-4 weeks of expansion in PL- supplemented media. The majority of CD90+FAP“ reticular fibroblasts were TGM2 negative. Overall, the data indicates a mixed early time point surface marker expression in fibroblast groups. A longer culture period led to a more homogenous population. The FBS to PL transition shifted the fibroblast phenotype over time, as cells acquired a more intermediate and papillary phenotype at the expense of reticular features.

[0133] A concentration range of 10-100% PP is able to produce a hydrogel after incubation with Thrombin for 1-2 h (Fig. 5). The gel that is formed is robust and can be readily handled when the concentration of PP is 25-100%. The rheological behaviour ofplatelet gel at concentrations of 100%, 25%, and 10% at 2 and 24 hr post-gelation was previously reported (Rahman et al 2021). A higher concentration of PP results in higher storage modulus and more effective cross-linking of the material regardless of hydrogel gelation time (24 h vs 2 h).

[0134] Cell counts were obtained and an Alamar Blue® assay was performed to measure the proliferation of cells. Representative confocal images for supplementation with FBS or PL are shown in Fig. 6. 2% PL supplementation in proliferation media supports fibroblast growth on the hydrogel at a similar rate to FBS supplementation (Fig. 7). Fibroblasts seeded on top (CT) on PG (24 h), divide more quickly than cells seeded within the hydrogel in both PL and FBS-supplemented growth media. Qualitative measurement of fibroblast infiltration through the hydrogel is summarised in Table 1. Overall, the best proliferation / gel infiltration was observed when fibroblasts were seeded on top of the hydrogel (range 10%-25% PP) at 24 hr post-gelation to assimilate a dermis-like tissue with fibroblasts present throughout the tissue. The 25% PP neodermis was easier to handle than the 10% PP neo-dermis.

[0135] Table 1 : Comparison of infiltration and survival of fibroblasts on top and within the hydrogel, day 6 post gelation

[0136] Cytokeratin pan-expression showed that fibroblasts seeded on top or within the hydrogel-supported epidermal stratification (Fig. 8). There was a trend in higher basement membrane deposition (shown by CollV expression) when fibroblasts were seeded on top of the hydrogel, but it did not reach significance (Fig. 8C). Conversely, there was a trend of reduced accumulation of differentiation marker, Keratin 10, in neo-epidermis, if fibroblasts were seeded on top of the hydrogel and, therefore, in close proximity to keratinocytes (Fig. 8D).

[0137] P-HSE proliferation depends on number of cells seeded (Fig. 9). The proliferation plots were a bell curve and peaked when fibroblasts range 0.25 x 106to 0.3 x 106 / cm2and keratinocytes range from 0.5 - 0.7 x 106 / cm2(Fig. 10). An outlier was observed for 0.5 x 106fibroblasts and 1 x 106keratinocytes / cm2. This was observed for all PL and FBS concentrations tested.

[0138] P-HSEs were stained for Cytokeratin 5 (CK5, a marker for stem and progenitor adult keratinocytes), Cytokeratin 10 (CK10, a marker for differentiating adult keratinocytes), PanCK, and Collagen IV (a marker for basement membrane at dermal- epidermal junction). Representative images are shown in Fig. 11. The stem and progenitor cell marker CK5 is expressed in P-HSE supplemented with both PL and FBS maturation media. Expression of CK5 in 0.1% PL-supplemented media had a bell distribution that was depended on the number of fibroblasts and keratinocytes seeded (Fig. 12A). P-HSEs formed with 0.2 - 0.3 x 106 / cm2fibroblasts and 0.4 - 0.6 x 106 / cm2keratinocytes cultured in 0.1 % PL-supplemented maturation media had the highest CK5 expression. An outlier was also observed with 0.45 x 106 / cm2fibroblasts and 0.9 x 106 / cm2keratinocytes. Overall, CK5 expression was the most stable for P-HSE matured in 0.1 % PL-supplemented media. CK5 expression was lower in 0.2% PL-supplemented maturation media, and there was variable expression observed in 0.3% PL- supplemented maturation media. Expression of the maturation marker CK10 was lower than CK5 but the trends in expression observed was similar to that observed for CK5 (Fig. 12B).

[0139] The presence of interfollicular stem and progenitor keratinocytes in P-HSE was confirmed with another marker, pi integrin / CD29, independently using immunofluorescence (Fig. 13). The identity of the fibroblasts was also confirmed using a Vimentin antibody. Unlike CK5 (Fig. 12A), there was a decrease in CD29 and Vimentin expression with a cell density of 0.25 x 106cells / cm2fibroblasts 1 0.5 x 106cells / cm2keratinocytes. Therefore, a functional analysis was required to identify optimal cell seeding density.

[0140] The P-HSE barrier function was tested using a LY assay (Fig.14). The data shows an inverted bell shape distribution of LY penetration, depending on the number offibroblasts / keratinocytes that were seeded (Fig. 14). P-HSEs with 0.2 x 106cells / cm2- 0.35 x 106cells / cm2fibroblasts and 0.4 x 106- 0.7 x 106keratinocytes showed the least LY penetration, and thus was identified as the optimal seeding range. P-HSE with 0.25 x 106cells / cm2fibroblasts and 0.5 x 106keratinocytes showed the least LY penetration and therefore had the greatest barrier function for both 0.1 % PL- and 0.3% FBS- supplemented maturation media on day 5 (i.e. on the day of grafting). Human native skin showed no LY penetration.

[0141] The barrier function was detected by confocal microscopy showing reduced LY penetration overtime on days 1 , 3 and 5 post keratinocytes seeding. Barrier function was compared to lack of LY penetration on native skin (Fig. 15). There was a gradual skin maturation, which resulted in the build-up of a barrier on the PL-supplemented serum-free 3D-engineered skin from days 1-5, shown by reduction of Lucifer yellow penetration into skin over time.

[0142] HSE composite structure with the maturation of both neo-dermis and neoepidermis over time is shown (Fig. 16). Days 1 - 3 were too early to form a stratified neo-epidermis. By day 8 - 11 , mature keratinocytes were losing their nucleus and the P-HSE was in a state of terminal differentiation. Day 5 was optimal for achieving a stratified neo-epidermis. PL induced stratification of keratinocytes and formed a dermal / epidermal double layer 3D structure, in a similar way to FBS at all cell densities tested (Fig. 16). The optimal cell seeding range to produce a live, mature skin graft that has some barrier function was determined by further analysis of the P-HSE. This method of preparing HSE can apply to both autologous and allogenic skin grafts.

[0143] The ultrastructure of P-HSE in fetal bovine serum (Fig. 17A) and serum-free (Fig. 17B) was analysed by TEM. 0.1% PL was able to support P-HSE architecture similar to 0.3% FBS. The basal keratinocytes had finger-like microvillae in both engineered skin (Norlen 2008). On the superficial layers of both engineered skin, the differentiated keratinocytes were flattened, gradually losing their chromatin. The basement membrane was less rigid in engineered skin than it was in native skin (Figure 17C). Consequently, basal keratinocytes lacked detectable hemidesmosomes which may reflect relatively free movement between dermis and epidermis through the hydrogel, compared to native collagen-based dermis. Fibroblasts are seemingly more active than their counterparts in native skin containing a large number of vesicles, Golgibodies and lipid droplets, typically observed in stimulated fibroblasts in culture (Gorin et al. 1982). Similar to native skin, some fibroblasts in engineered skin lie horizontally underneath the basement membrane maximising their surface towards basal keratinocytes. Desmosomes (attached to keratin intermediate filaments) were abundant between keratinocytes in engineered skin.

[0144] A cross-sectional view of the P-HSE structure by SEM on day 5, shows similar pore size in neo-dermis in 0.1% PL (average pore area 50.84 pm2) and 0.3% FBS (average pore area 58.96 pm2) maturation media (Fig. 18). Porous neo-dermis is covered by neo-epidermis in both serum-free (9.65 + 0.5 pm thickness) and FBS (8.69 + 0.5 pm thickness) supplemented maturation media. The top view of the skin confirmed presence of a mature epidermis, similar to native skin, with little detectable pores on the surface.

[0145] Skin maturation was mediated, at least partly, by TGF-pi (Fig. 19). This was shown by incubating the serum-free 3D-engineered skin in presence of TGF-pi neutralising antibody. The number of proliferative cells was identified by immunofluorescence using the Ki67 antibody. It was shown that proliferative cell numbers were significantly increased in presence of TGF-pi neutralising antibody, compared to the non-specific antibody. There was significantly more proliferation in serum-free 3D skin in presence of TGF-pi neutralising antibody compared to the isotype control antibody. The results for 3D-engineered skin that was cultured in 0.3% FBS or 0.1% PL -supplemented maturation media were comparable.

[0146] Both P-HSEs that were matured in 0.1% PL or 0.3% FBS-supplemented media before grafting formed a continuous thick epidermis over the full thickness wound (Fig. 20). The wound contraction was measured in grafts over time. P-HSE grafts that had been matured in PL performed better than P-HSEs matured in FBS, as they caused less wound contraction 14 days post grafting, compared to P-HSE matured in FBS.

[0147] 14 days post-graft, the human-specific keratinocyte differentiation marker involucrin was detected using immunofluorescence (Fig. 21A). Stem and progenitor keratinocytes marked with Integrin alpha6 (Fig. 21 B) and Integrin beta 1 (CD29, Fig. 21C) are also present 14 days post graft. They were observed at similar levels in grafts matured in 0.3% FBS or 0.1% PL.

[0148] Masson’s trichrome stain binds to collagen fibers and CD31 is a marker that is highly expressed on the surface of endothelial walls, and thus is used to determine vascularisation. P-HSE matured in 0.1% PL- or 0.3% FBS-supplemented media had similar levels of vascularisation and collagen deposition compared to autologous full thickness grafts in NUDE mice. There were similar levels of vascularisation and collagen deposition in 0.1 % P-HSE compared to an autologous full thickness graft (Fig. 22).

[0149] The barrier function of P-HSE in grafted mice 2 weeks post-graft was analysed (Fig. 23). Similar water retention was observed in P-HSE grafts compared to autologous full thickness graft and native mouse skin 14 days post-graft, indicating a fully mature, functional skin graft.

[0150] In summary, both PL and FBS supplements can support 2D fibroblast expansion (2% PL is comparable to 4% FBS) and 3D skin composite maturation (0.1% PL is comparable to 0.3% FBS) when using a platelet derived hydrogel as the scaffold. Best structural and functional P-HSE is obtained when the hydrogel is set for 24 h, followed by seeding the fibroblasts and keratinocytes on top of the hydrogel in a sequential manner, 24 h apart. We also identified the optimum fibroblast and keratinocyte cell density on the hydrogel as 0.25 x 106cells / cm2and 0.5 x 106cells / cm2, respectively. The P -HSE prepared in this manner, was ready for grafting in 5-7 days. P- HSE closed full thickness wound in mice shown by both histological and functional analysis.References

[0151] Banakh I., et al., A comparative study of engineered dermal templates for skin wound repair in a mouse model. Int J Mol Sc / , 2020. 21 (12)

[0152] Bayer, A., et al., Platelet-Released Growth Factors Induce Differentiation of Primary Keratinocytes. Mediators Inflamm, 2017. 2017: p. 5671615.

[0153] Berndt, S., et al., Autologous Platelet-Rich Plasma (CuteCell PRP) Safely Boosts In Vitro Human Fibroblast Expansion. Tissue Eng Part A, 2019. 25(21-22): p. 1550-1563.

[0154] Boyce, S.T., Methods for the Serum-Free Culture of Keratinocytes and Transplantation of Collagen-GAG-Based Skin Substitutes. Methods Mol Med, 1999. 18: p. 365-89.

[0155] Dimri, G.P., et al., A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proc Natl Acad Sci U S A, 1995. 92(20): p. 9363-7.

[0156] Dunn, L., et al., Murine model of wound healing. J Vis Exp, 2013(75): p. e50265.

[0157] Gorin, E., Gonen, H., & Dickbuch, S., A serum protein inhibitor of acid lipase and its possible role in lipid accumulation in cultured fibroblasts. Biochem J, 1982. 204(1), 221-227

[0158] Jafar, H., et al., Platelet lysate promotes the healing of long-standing diabetic foot ulcers: A report of two cases and in vitro study. Heliyon, 2020. 6(5): p. e03929.

[0159] Norlen, L. Exploring skin structure using cryo-electron microscopy and tomography. Eur J Dermatol, 2008, 18(3), 279-284.

[0160] O'Callaghan, N.J. and M. Fenech, A quantitative PCR method for measuring absolute telomere length. Biol Proced Online, 2011. 13: p. 3.

[0161] Rahman, M.M., et al., A platelet-derived hydrogel improves neovascularisation in full thickness wounds. Acta Biomater, 2021. 136: p. 199-209.

[0162] Sierra-Sanchez, A., et al., Cellular human tissue-engineered skin substitutes investigated for deep and difficult to heal injuries. NPJ Regen Med, 2021. 6(1): p. 35.

Claims

CLAIMS1. A method for preparing an artificial skin tissue comprising:- expanding fibroblast and keratinocyte cells from a subject, wherein the fibroblast cells are expanded in serum-free proliferation media comprising platelet lysate,- preparing a platelet-derived hydrogel from platelet precipitate,- seeding the cells within or on the platelet-derived hydrogel to form the artificial skin tissue, and- cultivating the artificial skin tissue in serum-free maturation media comprising platelet lysate.

2. The method according to claim 1, wherein the serum-free proliferation media comprises about 0.5% to about 4% PL.

3. The method according to claim 1 or 2, wherein the serum-free maturation media comprises about 0.1% to about 0.5% PL.

4. The method according to any one of claims 1-3, wherein the serum-free maturation media, the serum-free proliferation media, or a combination thereof comprises: transforming growth factor pi (TGF-pi) neutralising antibody, lgG1 , or combinations thereof.

5. The method according to any one of claims 1-4, wherein the hydrogel is prepared using platelet precipitate (PP) at a concentration of from about 10 - 25%.

6. The method according to any one of claims 1-5, wherein the fibroblast cells are seeded at a concentration of about 0.15 - 0.5 x 106 / cm2.

7. The method according to any one of claims 1-6, wherein the keratinocyte cells are seeded at a concentration of about 0.3 - 1 x 106 / cm2.

8. The method according to any one of claims 1-7, wherein the artificial skin tissue is cultivated for about 2 - 7 days.

9. The method according to any one of claims 1-8, wherein the cells are seeded on the hydrogel after gelation of the hydrogel, thereby providing cells disposed on an outer surface of the hydrogel to form the artificial skin tissue.

10. The method according to claim 9, wherein seeding the cells on the hydrogel comprises a first step comprising seeding the fibroblast cells on the hydrogel, and a second step comprising seeding the keratinocyte cells on the hydrogel.

11. The method according to any one of claims 1-8, wherein- the fibroblast cells are seeded within the hydrogel during gelation of the hydrogel, thereby providing fibroblast cells embedded within the hydrogel, and- the keratinocyte cells are seeded on the hydrogel to form the artificial skin tissue.

12. An artificial skin tissue prepared by the method of any one of claims 1-11.

13. A method of treating a dermatological disease or symptom comprising contacting diseased or loss of skin with the artificial skin tissue of claim 12.

14. The method of claim 13, wherein the diseased or loss of skin comprises a skin wound.

15. The method of claim 14, wherein the artificial skin tissue promotes closure of the skin wound.

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