WOUND HEALING FORMULATION

Abstract

The present disclosure relates to a method for producing a composition, wherein the method comprises the steps of co-culturing immortalized fibroblasts and immortalized keratinocytes, thereby producing secretion; separating the secretion from the fibroblasts and keratinocytes; and providing a pharmaceutically acceptable composition comprising the secretion. The present disclosure also relates to the composition obtainable by the method, wherein the composition preferably is a pharmaceutical composition for medical use, preferably for use in the treatment of a wound, preferably a chronic or acute wound.

Claims

1. Method for producing a therapeutic wound healing composition, wherein the method comprises the steps of: a) co-culturing immortalized fibroblasts and immortalized keratinocytes, thereby producing secretion; b) separating the secretion from the fibroblasts and keratinocytes; and c) providing a therapeutic wound healing composition comprising the secretion.

2. Method according to claim 1, wherein in step a) the percentage of the immortalized fibroblasts relative to the total number of immortalized fibroblasts and immortalized keratinocytes is between 5% and 95%.

3. Method according to of claims 1, wherein the immortalized fibroblasts are TERT-immortalized fibroblasts; and/or the immortalized keratinocytes are A431 keratinocytes or TERT-immortalized keratinocytes.

4. Method according to claims 1, wherein step a) is performed in vitro; at 30-40 C.; for at least 1 hour; and/or in a clinical grade medium.

5. Method according to claim 1, wherein in step a) the immortalized fibroblasts and immortalized keratinocytes are arranged randomly in the co-culture; and/or the immortalized fibroblasts and immortalized keratinocytes are co-cultured together in a monolayer of cells.

6. A composition obtainable by the method according to claim 1, wherein the composition is a pharmaceutically acceptable composition.

7. A method for the treatment of a wound, comprising applying the composition of claim 6 to the wound in an amount effective for the treatment of the wound.

8. The method according to claim 7, wherein the treatment of a wound comprises applying the composition onto a skin wound.

9. The composition according to claim 6, wherein the composition contains no fibroblasts and/or keratinocytes.

10. The composition according to claim 6, wherein the composition is in the form of a liquid, ointment, cream, gel, hydrogel, lotion, dressing, or carrier.

11. Method according to claim 1, wherein co-culturing immortalized fibroblasts and immortalized keratinocytes, thereby producing secretion, provides a percentage of the immortalized fibroblasts relative to the total number of immortalized fibroblasts and immortalized kerotinocytes between 25% and 95%.

12. Method according to claim 2, wherein the immortalized fibroblasts are TERT-immortalized fibroblasts; and/or the immortalized keratinocytes are A431 keratinocytes or TERT-immortalized keratinocytes.

13. Method according to claim 4, wherein co-culturing immortalized fibroblasts and immortalized keratinocytes, thereby producing secretion, is performed at 34-38 C.

14. Method according to claim 4, wherein co-culturing immortalized fibroblasts and immortalized keratinocytes, thereby producing secretion, is performed for at most 240 hours.

15. Method according to claim 1, wherein co-culturing immortalized fibroblasts and immortalized keratinocytes, thereby producing secretion, is performed in vitro; at 34-38 C.; for 1 hour to 240 hours; and in a clinical grade medium.

16. The method of claim 8, wherein the applying the composition onto a skin wound is followed by applying a dressing, compression therapy, or a tissue substitute or an autograft onto the skin wound.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0077] FIG. 1. Flow diagram of production of the Wound Healing Formulation (WHF)

[0078] FIG. 2A. A fibroblast scratch assay shows a remarkable reduction in scratch size after 2 and 3 days with a 10% dilution of the WHF as compared to longEGF (1 ng/ml) alone and the negative control. Briefly, primary fibroblasts were cultured in a monolayer until 70% confluency, followed by a 4-days serum free culture period. A scratch was made which represents a wound. After wounding different culture conditions were added: 10% WHF TERT fib/A431 (75%/25%) supernatant (filled squares), longEGF (1 ng/ml) (dotted line) and a negative control (CKC1 medium only without supplements)(open squares). After 2 days the cultures were refreshed with similar culture conditions. Within the graph the relative reduction in scratch size is shown (n=4 donors, quadruplo, MEANSEM). The curves were statistically different as measured with a 2-way ANOVA followed by Tukey's multiple comparisons test.

[0079] FIG. 2B. Keratinocyte migration assay. Epidermal sheets from foreskin of 3 donors (duplo) were placed on a acellular donor dermis. After attachment the cultures were cultured air-exposed with different conditions (10% WHF, 30% WHF (both derived from TERT fib/A431 (75%/25%), longEGF (1 ng/ml) and medium only (neg. CKC2, including actrapid, isoprenaline and solucortef)) for approximately 2 weeks. The epidermal outgrowth was measured on histological H&E stained sections with NIS Elements. The graph shows a significant increased epidermal outgrowth which is stimulated by the WHF (10% and 30%). To check whether the longEGF is working, we added results from another experiment in which a higher concentration (2 ng/ml) and longer air-exposed culture period (3 wk) was used. As shown in Figure, after two weeks of air-exposed culture, the outgrowth of the epidermal sheets treated with 10% or 30% WHF was superior to longEGF (1 ng/ml) alone and the negative control. Data are represented as MEANSEM. Statistical analysis: one-way ANOVA; followed by Tukey's multiple comparisons test. * P<0.05; **P<0.01; ***P<0.001; ****P<0.0001

[0080] FIG. 3. Following the keratinocyte migration assay, H&E staining of the outgrowth shows that the epidermal outgrowth of the WHF supernatants (10% and 30%, derived from TERT fib/A431 (75%/25%)) indeed exhibits a better quality than the negative control and longEGF (1 ng/ml).

[0081] FIG. 4. Proliferation rate and viability of primary fibroblasts and keratinocytes was determined in the presence of 10% WHF, 30% WHF (both derived from TERT fib/A431 (75%/25%)), bFGF, EGF, or negative control. Briefly, primary fibroblasts (3.84*10.sup.4 cells/well, dy0 and dy4) or primary keratinocytes (2.24*10.sup.5 cells/well, dy0 and 2.0*10.sup.5 cells/well, dy3) were seeded into a 6-wells plate. Cells were counted with the Adam cell counter which also recorded the cell viability. Culture conditions: 10% and 30% WHF supernatant (TERT fib/A431 (75%/25%)). Positive control for fibroblasts was bFGF (10 ng/ml) and for keratinocytes EGF (10 ng/ml) and normal medium was used as negative control. When the cells reached 70-80% confluency, they were passaged, counted and cultured again for the same period. The primary fibroblasts and keratinocytes derived from normal skin (n=2) and foreskin (n=1). As can be seen in the Figure, WHF (10% and 30%) does not significantly affect the proliferation and viability of both cell types, however there is a positive trend of the WHF. Values represent MEANSEM, 3 donors (duplo).

[0082] FIG. 5. Differences in wound healing potency of the secretion of the cell line co-cultures (75/25, i.e. (75% TERT fib/25% A431)) compared to the secretion of the primary cell counterpart derived from either foreskin or adult abdominal skin (75% primary dermal fibroblasts/25% primary epidermal keratinocytes). 24 hour supernatant ELISA (IL-6 and IL-8) measurements were performed for the cocultures. Values represent MEANSEM.

[0083] The following Examples illustrate different embodiments of the disclosure. Unless stated otherwise all recombinant DNA techniques are carried out according to standard protocols as described in e.g. Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press; and Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, NY; and in Volumes 1 and 2 of Ausubel et al. (1994) Current Protocols in Molecular Biology, Current Protocols, USA.

EXAMPLE 1

[0084] In order to determine the endogenous secretion of three different cell lines, i.e. A431 keratinocytes, TERT-keratinocytes and BJ5-ta fibroblasts, 24-hour supernatants were collected from 70-80% confluent monolayer cell line cultures for ELISA measurements, see Table 2 below (n=2-3 experiments, duplo). Cell lines were cultured within clinical grade medium (CKC1) and for the production of secretion for 24 hours in clinical grade DMEM/HamF12 (3:1) medium only.

TABLE-US-00002 TABLE 2 Secretion of the three different cell lines when cultured separately. Concentration in pg/ml after 24 hours of culturing (MEAN SD), ND = below detection limit. A431 TERT keratinocytes keratinocytes BJ5-ta fibroblasts CCL-2/ ND ND 5.957 1.712 MCP-1 CXCL-1/ 61.605 63.374 ND 698 21 GRO CXCL-8/ 24.168 26.919 716 161 2.227 1.762 IL-8 CCL-5/ 1.198 421 ND 34 RANTES IL-6 129 ND 275 215 IL-1 173 85 ND ND TNF- ND ND ND TIMP-2 11.537 5.356 22.834 2.566 157.043 67.116 VEGF 53.018 12.985 2518 546 255 68 HGF ND ND 1.732

EXAMPLE 2

[0085] This example shows that the secretion of a co-culture of immortalized keratinocytes and immortalized fibroblasts is richer in wound healing mediators as compared to the secretion of the cell lines when cultured separately (as in Example 1), and also as compared to a full-thickness skin equivalent or excised skin.

[0086] Multiple immortalized keratinocyte cell lines were tested (i.e. A431 obtainable from ATCC, CRL-1555; N/TERT-1 obtainable from Rheinwald's lab; and NCTC2544 obtainable from Italy, Cell Culture center, Istituto Zooprofilattico sperimentale; BS CL143) in a co-culture with the TERT-immortalized fibroblast cell line BJ-5ta (obtainable from ATCC, CRL-4001), wherein different ratios between keratinocytes and fibroblasts were used. The combination of the A431 keratinocyte cell line and the TERT fibroblast cell line in a 25%/75% ratio resulted in the most potent secretion (Wound Healing Formulation, WHF). Below, the experimental procedure and results are described in more detail.

[0087] Human Cell Line Skin Equivalent

[0088] To establish the interaction between the keratinocytes and fibroblasts, first a human skin equivalent was successfully constructed from immortalized keratinocytes and immortalized fibroblasts. This full-thickness cell line skin equivalent consisted of a well-differentiated epidermis on top of a fibroblast populated dermis. A haematoxylin-eosin staining of the skin equivalent showed a stratified epidermal layer with a stratum basale, stratum spinosum, stratum granulosum and stratum corneum. Within the dermal layer the TERT fibroblasts are present and produce their own matrix. A basement membrane is formed by interaction between the TERT immortalized keratinocytes and fibroblasts.

[0089] This skin equivalent was able to secrete a reasonable amount of multiple wound-healing mediators (like IL-6; CXCL-1/IL-8; CCL-2/MCP-1; CXCL-1/GRO-; RANTES/CCL-5; VEGF; TIMP-2 (Table 3 below).

[0090] Co-Culture

[0091] Surprisingly, a more simplified cell culture model (see FIG. 1), i.e. a co-culture of both immortalized keratinocytes and immortalized fibroblasts in a monolayer resulted in substantially higher wound-healing mediator levels than the secretion of a full-thickness skin equivalent (Table 3). Multiple immortalized keratinocyte cell lines were tested (A431, TERT keratinocytes and NCTC2544) in a co-culture with TERT-immortalized fibroblasts using different ratios.

[0092] The cell lines co-cultures were first cultured within clinical grade medium until 70% confluency was reached. For secretion production, the co-cultures were then cultured for 24 hours in clinical grade DMEM/HamF12 (3:1) medium only (obtainable from e.g. Sigma Aldrich).

[0093] After collection of 24 hour supernatant, ELISA measurements were performed on the secretions of different subsets of cultures. The human cell line equivalent was able to produce a cocktail of wound-healing mediators. In comparison with the ex vivo skin, the protein levels were either similar, increased or decreased. However, the co-cultures resulted in a much more enriched mixture of wound-healing mediators. Both the combination of TERT fibroblasts/A431 (75%/25%) and TERT fibroblast/TERT keratinocytes (75%/25%) resulted in a very potent wound-healing mediator cocktail, especially the combination of TERT fibroblasts (75%) and A431 keratinocytes (25%), see Table 3. Values are represented as MEANSD. ND=below detection limit.

TABLE-US-00003 TABLE 3 Secretion of the two different cell line cocultures is richer in chemokines, cytokines and proteases & growth factors as compared to secretion of ex vivo skin, or secretion of a human cell line skin equivalent. Concentration in pg/ml after 24 hours of culturing (MEAN SD), ND = below detection limit. Cell line skin equivalent Co-culture Co-culture TERT fib/TERT kera TERT fib/TERT kera TERT fib/A431 Ex vivo skin (R&D medium) (75/25) (75/25) CCL-2/MCP-1 1.497 1.124 15.945 7.442 64.333 18.457 107.451 51.257 CXCL-1/GRO 3.109 1.689 35.439 17.009 55.901 36.781 153.556 113.443 CXCL-8/IL-8 19.647 13.512 22.655 12.774 250.964 138.051 349.466 146.296 CCL-5/RANTES 83 40 108 16 148 61 2.061 843 IL-6 131.756 64.057 568 315 13.987 3.617 18.924 4.741 IL-1 22 15 ND ND 82 TNF- ND ND ND ND TIMP-2 38.355 32.576 35.010 5.846 93.238 36.037 65.148 18.320 VEGF 2.425 499 ND ND 7.011 793 HGF 6.227 4.209 ND 447 308

[0094] Upscaling from the regular T75 culture flasks towards a Nunc Factory system with 2 layers, suitable for high yield and large scale production with lot-to-lot consistency, was successful. Since the co-culture TERT fibroblasts in combination with A431 resulted in the most potent cocktail, it was decided to proceed with those two cell lines. Direct filtering (before 20 C. storage) of the supernatant with a 0.22 m filter appeared to have no effect on the protein concentration.

EXAMPLE 3

[0095] This example describes the differences in wound healing potency of the secretion of different co-cultures, wherein different ratios between fibroblasts and keratinocytes were applied. As described earlier, the 24 hour supernatant ELISA measurements were performed not only on different co-cultures, i.e. TERT fibroblasts either combined with A431 or TERT keratinocyte cell line, but also on cocultures having different ratios of fibroblasts/keratinocytes (i.e. 0/100; 25/75; 50/50; 75/25; 100/0). The results thereof are shown in Tables 4 and 5.

[0096] Most enriched was the secretion of a co-culture comprising 75% TERT fibroblasts and 25% A431 or TERT keratinocytes (Table 4). As already described in Example 1 and 2, the cell lines co-cultures were first cultured within clinical grade medium (CKC1) until they reached 70% confluency. For secretion production, the cell line co-cultures were cultured for 24 hours in clinical grade DMEM/HamF12 (3:1) medium only.

TABLE-US-00004 TABLE 4 Secretion of coculture of TERT fibroblasts and A431 keratinocytes in different ratios. Concentration in pg/ml after 24 hours of culturing (MEAN SD), ND = below detection limit. Ratio: Ratio: Ratio: Ratio: Ratio: 0/100 25/75 50/50 75/25 100/0 CCL-2/MCP-1 ND 7.945 8.313 42.488 43.570 107.451 51.257 6.151 1.758 CXCL-1/GRO 61.605 63.374 98.511 87.228 120.028 90.241 153.556 113.443 732 CXCL-8/IL-8 24.168 26.919 119.176 59.049 265.165 17.477 349.466 146.296 1.860 1.480 CCL-5/RANTES 1.198 421 2.284 730 2.548 1.001 2.061 843 68 IL-6 129 2.517 1.715 8.270 2.659 18.924 4.741 267 197 IL-1 173 85 119 34 87 66 82 ND TNF- ND ND ND ND ND TIMP-2 11.537 5.356 25.058 10.379 37.710 17.754 65.148 18.320 170.096 77.132 VEGF 53.018 12.985 42.614 8.541 27.099 4.988 7.011 793 261 46 HGF ND ND ND 308 1.865

TABLE-US-00005 TABLE 5 Ratio TERT fibroblasts/TERT keratinocytes and resulting concentration wound healing mediators in the secretion (pg/ml) after 24 hours of culturing (MEAN SD), ND = below detection limit. Ratio: Ratio: Ratio: Ratio: Ratio: 0/100 25/75 50/50 75/25 100/0 CCL-2/MCP-1 ND 12.221 10.075 33.018 24.192 64.333 18.457 5.763 1.666 CXCL-1/GRO ND 30.804 42.189 50.580 59.793 55.901 36.781 664 42 CXCL-8/IL-8 716 161 95.121 73.078 223.312 54.284 250.964 138.051 2.593 2.043 CCL-5/RANTES ND 147 19 150 45 148 61 ND IL-6 ND 2.787 2.201 9.258 4.768 13.987 3.617 283 233 IL-1 ND ND ND ND ND TNF- ND ND ND ND ND TIMP-2 22.834 2.566 46.476 20.943 73.923 26.893 93.238 36.037 143.991 57.100 VEGF 2518 546 563 ND ND 248 89 HGF ND ND ND 447 1.598

EXAMPLE 4

[0097] This example describes different wound healing assays that were performed. The secretion (Wound Healing Formulation, WHF) derived from the TERT fibroblast/A431 (75%/25%) co-culture was tested in different in vitro test models using primary fibroblasts and keratinocytes from different donors as proof of principle.

[0098] First, the effect of the WHF was tested on the migration of fibroblasts. Within a fibroblast scratch assay a remarkable reduction in scratch size was observed after 2 and 3 days with a 10% dilution of the WHF as compared to longEGF (1 ng/ml) alone and the negative control (FIG. 2A). Briefly, Primary fibroblasts were cultured in a monolayer until 70% confluency, followed by a 4-days serum free culture period. A scratch was made which represents a wound. After wounding different culture conditions were added: 10% WHF TERT fib/A431 (75%/25%) supernatant (filled squares), longEGF (1 ng/ml) (dotted line) and a negative control (CKC1 medium only without supplements)(open squares). After 2 days the cultures were refreshed with similar culture conditions. Within the graph the relative reduction in scratch size is shown (n=4 donors, quadruplo, MEANSEM). The curves were statistically different as measured with a 2-way ANOVA followed by Tukey's multiple comparisons test.

[0099] Secondly, the effect of the WHF on the migration behaviour of keratinocytes was established (FIG. 2B). Briefly, the epidermal sheets from foreskin of 3 donors (duplo) were placed on a acellular donor dermis. After attachment the cultures were cultured air-exposed with different conditions (10% WHF, 30% WHF, longEGF (1 ng/ml) and medium only (CKC2, including actrapid, isoprenaline and solucortef) for approximately 2 weeks. The epidermal outgrowth was measured on histological H&E stained sections with NIS Elements. The graph shows a significant increased epidermal outgrowth which is stimulated by the WHF (10% and 30%). To prove that the longEGF is working, we added results from another experiment in which a higher concentration (2 ng/ml) and longer air-exposed culture period (3 wk) was used. As shown in FIG. 2B, after two weeks of air-exposed culture, the outgrowth of the epidermal sheets treated with 10% or 30% WHF was superior to longEGF (1 ng/ml) alone and the negative control. In addition, the quality of the restored epidermis was better than long EGF (1 ng/ml) and the negative control. As can be seen in FIG. 3, the H&E staining of the outgrowth shows that the epidermal outgrowth of the WHF supernatants (10% and 30%) indeed exhibits a better quality than the negative control and longEGF (1 ng/ml).

[0100] Thirdly, the effect of the WHF on normal proliferation rate and viability of primary fibroblasts and keratinocytes was determined. Briefly, primary fibroblasts (3.84*10.sup.4 cells/well, dy0 and dy4) or primary keratinocytes (2.24*10.sup.5 cells/well, dy0 and 2.0*10.sup.5 cells/well, dy3) were seeded into a 6-wells plate. Cells were counted with the Adam cell counter which also recorded the cell viability. Culture conditions: 10% and 30% WHF supernatant (TERT fib/A431 (75%/25%)). Positive control for fibroblasts was bFGF (10 ng/ml) and for keratinocytes EGF (10 ng/ml) and normal medium was used as negative control. When the cells reached 70-80% confluency, they were passaged, counted and cultured again for the same period. The primary fibroblasts and keratinocytes derived from normal skin (n=2) and foreskin (n=1). As can be seen in FIG. 4, WHF (10% and 30%) does not significantly affect the proliferation and viability of both cell types, however there is a positive trend of the WHF. Values represent MEANSEM, 3 donors (duplo).

[0101] In summary, within a fibroblast scratch assay, a 10% dilution of WHF showed to reduce scratch size more than long EGF (1 ng/ml) alone. Within a keratinocyte migration assay, 10% and 30% dilutions of WHF stimulated epidermal outgrowth more as compared to longEGF (1 ng/ml) alone. In addition, WHF does not affect proliferation and viability of the primary fibroblasts and primary keratinocytes used in the assays. In conclusion, the WHF is able to improve wound-closure as shown by the in vitro migration studies, while maintaining the normal proliferation rate and viability of the cells.

EXAMPLE 5

[0102] A coculture of immortalized keratinocytes and immortalized fibroblasts can produce a richer secretion as compared to a coculture of primary keratinocytes and primary fibroblasts. This example describes the differences in wound healing potency of the secretion of the immortalized cell line co-cultures (75/25) compared to the secretion of the primary cell counterpart derived from either foreskin or adult abdominal skin (75% primary dermal fibroblasts/25% primary epidermal keratinocytes). As described earlier, 24 hour supernatant ELISA (IL-6 and IL-8) measurements were performed for the cocultures (see FIG. 5)

[0103] Notably, most enriched was the secretion of a co-culture comprising 75% TERT fibroblasts and 25% A431 (cell line). As already described in Example 1 and 2, the co-cultures were first cultured within clinical grade medium (CKC1) until they reached 70% confluency. For secretion production, the co-cultures were cultured for 24 hours in clinical grade DMEM/HamF12 (3:1) medium only.