REJUVENATION FIELD OF TECHNOLOGY

The present invention relates to a method for producing a dermal fibroblast conditioned sera, and a skin rejuvenation composition derived therefrom. More particularly, the sera and its composition are able to promote re-epithelialization of keratinocytes that leads to wound closure.

BACKGROUND OF THE INVENTION

Skin is important as a first line defence against environmental and pathogenic attack. Anatomically, skin tissue consists of two layers: the superficial epidermal layer which is mainly made up of keratinocyte and the deeper dermal layer which is made up of fibroblast. Skin tissue has ability to regenerate or self-renewal upon injury by activating wound healing process. Wound healing is a complex process, which have overlapping stages including haemostasis, inflammation, proliferation or granulation, and remodelling or maturation. In wound healing, epithelialization, fibroplasias and angiogenesis occur in proliferative phase while collagen formation occur during maturation phase. Once injury occurs, paracrine signals stimulate fibroblasts to secrete wound healing mediators, so that the epithelial cells actively proliferate, migrate, and differentiate to regenerate the damaged tissue. Wound healing was impaired in aging, disease cases as well as all cases of chronic wounds due to lack of growth factors and extracellular matrix protein. In elderly individual, every phase of healing undergoes characteristic of age-related changes, including decreased secretion of growth factors, reduced collagen turnover and remodelling, enhanced platelet aggregation, collagen deposition, decreased wound strength, impaired macrophage function, delayed infiltration of macrophages and lymphocytes and increased secretion of inflammatory mediators. During healing process, it involves interaction of variety of cells, proteins, cytokines, growth factors and angiogenic factors. These factors can be collected from conditioned medium of cultured human or animal fibroblasts as dermal fibroblast sera and have a potential to become supplements for rejuvenation and for the treatment of skin injury. Conditioned medium is a formulation containing extracellular protein(s) and cellular metabolites, which supports the growth of any desired eukaryotic cell type, said cells having been cultured in either two or three dimensions. To achieve this goal, different approaches to producing dermal fibroblast sera, also known as dermal fibroblast conditioned medium, are studied and established.

The production and use of conditioned medium from mesenchymal stem cell (MSCs) culture is described in WO 2013121427 Al. The methods comprise culturing MSCs in a medium comprising nicotinamide or nicotinamide and fibroblast growth factor 4 (FGF4) to collect the conditioned medium. Compositions comprising the mesenchymal stem cell conditioned medium shown to promote wound healing.

The other process for preparing a conditioned cell culture medium is described in US 20120207705 Al. The method comprises a) culturing eukaryotic cells in a growth medium having a composition effective to support cell growth; b) separating the cultured cells from the growth medium; and c) maintaining the cultured cells in a basal medium having a composition suitable to maintain cell viability, but not to support substantial cell growth. The cells are preferably dermal sheath, dermal papilla or dermal fibroblast cells. The compositions are useful as pharmaceutical compositions, especially for wound healing. Both aforementioned prior arts are mentioned methods for preparing conditioned medium and show its potential in wound healing focusing on remodelling phase. It would, therefore, be advantageous to produce conditioned medium that can facilitate epithelial regeneration, repair, healing i.e. re-epithelialization. In the present invention, secreted proteins from fibroblasts as dermal fibroblasts sera were produced that enhance epithelial proliferation and migration, thus potential to be used for skin rejuvenation.

SUMMARY OF THE INVENTION

One aspect of the present invention is to provide a culture medium which serves as a precursor of a composition for skin regeneration, more particularly it -is a conditioned culture medium capable of promoting re-epithelialization in terms of cell attachment, proliferation, and migration.

Another aspect of the present invention is to provide a conditioned culture medium which supports in vitro cultivation of keratinocytes.

Another aspect of the present invention is to provide a culture medium which serves as a precursor of a composition for skin rejuvenating, more particularly useful in repairing physical anomalies and cosmetic defects.

Another aspect of the present invention is to provide a composition suitable for topical applications. The composition may be formulated or used in association with a suitable pharmaceutical acceptable or cosmetic carriers. For example, the composition can be encapsulated in a hydrogel, or micro carriers for sustained release or delivery.

Physical embodiments of the composition include, but are not limited to, liquid or solid, frozen, lyophilized or dried into a powder. Also, the composition may be further processed to concentrate or reduce one or more components contained therein.

At least one of the preceding objects is met, in whole or in part, by the present invention, in which one of the embodiments of the present invention is a method of preparing a conditioned cell culture medium, also known as 'dermal fibroblast sera (DFS)' hereinafter, comprising the steps of: culturing dermal fibroblast cells in a culture medium comprising a serum-free keratinocyte-specific medium supplemented with stimulatory factors until the · medium contains fibroblast secreted proteins of fibronectin, serum albumin, collagen alpha- 1 (VI) chain, collagen alpha- 1(1) chain decorin, serotransferrin, pentraxin-related protein PTX3 and metalloproteinase inhibitor 1.

In the preferred embodiment, the method further comprises a step of filtering- the conditioned medium through a filter membrane having a cut-off size of 3 kDa. Preferably, the stimulatory factors are bovine pituitary extract (BPE), recombinant human insulin-like growth factor-I, hydrocortisone, bovine transferrin, basic fibroblast growth factor (bFGF), and/or human epidermal growth factor (EGF).

At least one of the preceding objects is met, in whole or in part, by the present invention, in which one of the embodiments of the present invention is a skin rejuvenation composition derived from the method as set forth in preceding description, more particularly, the composition is able to improve attachment, growth and migration of epithelial cells, thus, regeneration and rejuvenation of skin tissue. BRIEF DESCRIPTION OF THE DRAWINGS

For a better understanding of the nature and objects of the invention, reference should be made to the following detailed description taken in connection with the accompanying drawings.

Figure 1 shows a schematic drawing of a method (10) of preparing a dermal fibroblast conditioned medium and the use (20) thereof for in vitro culturing of epithelial cells. Figure 2 are micrographs taken by Light Microscopy (under magnification of 4x) showing the growth of epithelial cells in (a) a keratinocyte-specific basal medium (KM); (b) a keratinocytes-specific medium supplemented with stimulatory factors KMS); and (c) the conditioned medium derived from the preferred embodiment in the presence of the basal medium (denoted as DFS-KM).

Figure 3 is a graphical representation of the correlation between concentration of the DFS and epithelial cell concentration. Figure 4 is a graphical representation of the correlation between concentration of the DFS and epithelial cell growth rate.

Figure 5 is a graphical representation of the correlation between concentration of the DFS and epithelial cell migration rate.

Figure 6 is a graphical representation of the correlation between concentration of the DFS and epithelial cell wound healing rate.

BRIEF DESCRIPTION OF THE INVENTION

This invention relates to a method of producing a medium conditioned using dermal fibroblasts in a defined keratinocyte-specific medium, and a skin rejuvenation composition derived thereof. Exemplary, non-limiting embodiments of the invention will be disclosed. However, it is to be understood that limiting the description to the preferred embodiments of the invention is merely to facilitate discussion of the present invention and it is envisioned that those skilled in the art may devise various modifications without departing from the scope of the appended claim. The present invention provides a method of producing a conditioned cell medium which encourages keratinocyte attachment, proliferation, and migration. In the present invention, the method comprising a step of culturing dermal fibroblast cells in a culture medium comprising serum-free keratinocyte-specific medium (KM) supplemented with stimulatory factors until the medium contains fibroblast secreted proteins including fibronectin, serum albumin, collagen alpha- 1 (VI) chain, collagen alpha- 1(1) chain, decorin, fibulin-1, serotransferrin, pentraxin-related protein PTX3 and metalloproteinase inhibitor 1. Medium conditioned by dermal fibroblasts contains a variety of fibroblasts secreted proteins, including but not limited to extracellular matrices, growth factors, cytokines and small molecules (organic compound that helps in regulate biological process, in which the compound having a molecular weight of less than 900 Daltons). In accordance to preceding description, components present in the dermal fibroblast-conditioned medium using KM of the present invention are listed in Table 1 in Example 1 based on results from LC-mass spectrometry.

In the preferred embodiment, the method of preparing the conditioned cell culture medium comprising the steps of: 1) culturing dermal fibroblast cells in a serum-containing medium until 90-100% confluence; 2) washing the fibroblasts with phosphate buffered saline (PBS) to remove excess medium; 3) culturing the washed dermal fibroblast in the serum-free KM supplemented with stimulatory factors for 24 to 72 hours at approximately 37°C under a 5% C0 ₂ so as to obtain a conditioned medium containing the desired fibroblast secreted proteins; and 4) removing the conditioned medium from the cultured cells. The cells can be cultured in monolayer, beads (i.e., two-dimensions) or, preferably, in three- dimensions. The cells can be derived from human or animal, more preferably from human to reduce the risk of adversarial immune response. More particularly, the cells can be derived from fresh human dermal fibroblasts. Cryopreserved fibroblasts can also be used. The stimulatory factors are bovine pituitary extract (BPE), recombinant human insulin-like growth factor-I, hydrocortisone, bovine transferrin, basic fibroblast growth factor (bFGF), and/or human epidermal growth factor (EGF). In the present invention, the KM used is preferably a defined medium to avoid batch-to- batch variation, more preferably, the KM used can be Epilife's basal medium, and supplement used can be Epilife's Defined Growth Supplement (EDGS),- or the like. Amino acids (both D- and L-amino acids) such as glutamine, lysine, leucine, and methionine also added into the medium. The serum-free medium further supplemented with sodium ions, phenol red and 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid (HEPES) as pH indicators and buffering agent, respectively. Ingredients such as antibiotics (penicillin, streptomycin, amphotericin B, and etcetera) can also be added to prevent contamination. Salts such as calcium chloride, copper sulfate, magnesium chloride and magnesium sulfate can also be added therein.

In accordance to preferred embodiment, the method further comprises a step of filtering the conditioned medium through a filter membrane having a cut-off size of 3 kD. In the preferred embodiment, the conditioned medium was concentrated using centrifugal filter membrane and was dialyzed (<1 kD) as to obtain a concentration of 1600-2000 μg/ml. The dialyzed medium was placed into a beaker containing PBS without antibiotic for overnight at 4°C, and stirred for a minimum of 2 hours before collected.

Once the conditioned medium is ready, it may be used in any state. Physical embodiments of the conditioned medium include, but are not limited to, liquid or solid, frozen, lyophilized or dried into a powder. The conditioned medium may be formulated or used in association with suitable pharmaceutical acceptable or cosmetic carriers to form a composition. For example, the medium, or its composition, can be encapsulated in a tissue scaffold, hydrogel, or micro carriers for sustained release or delivery. Also, the medium, or its composition, may be further processed to concentrate or reduce one or more components contained therein. In the preferred embodiment, the conditioned medium can be used as-a main ingredient in a skin rejuvenation composition, such as to be incorporated into a lotion, toner, or a mask composition.

In another embodiment of the invention, the composition may be used to culture cells. The conditioned cell media of the invention contains factors useful in promoting cell attachment and growth. EXAMPLE

Exemplary method of the preferred embodiment is disclosed hereinafter. The present invention may be embodied in other specific forms without departing from its essential characteristics. The described embodiments are to be considered in all aspects only as illustrative and not restrictive. The scope of the invention is, therefore indicated by the appended claims rather than by the foregoing description. All changes, which come within the meaning and range of equivalency of the claims, are to be embraced within their scope.

EXAMPLE 1

Isolation of cells and primary culture

Skin samples were collected as redundant tissue from consenting patients before surgery. The skin was cleaned and washed with Dulbecco's phosphate buffered saline (DPBS; Gibco, USA) before being cut into small pieces. Then, skin was digested using 0.6% collagenase type I (Worthington, USA) solution in a shaker incubator for 4-5 H at 37°C, and subsequently treated with trypsin-EDTA (TE; Worthington) for 10 minutes, followed by neutralization with a trypsin inhibitor (Worthington). Digested tissues containing both fibroblasts and keratinocytes were centrifuged, and the pellet was suspended in co-culture medium (1 :1 mixture of F12:DMEM, FD; Gibco) with 10% fetal bovine serum (FBS; Gibco) and Epilife with growth supplement (Gibco). The cells were seeded into 6-well plates (Greiner Bio-one, USA) and grown at 37°C in 5% C0 ₂ . The culture medium was replaced every two days until cells reached 80%-90% confluence. Subsequently, co- cultured cells were treated with 0.05% TE for 5 minutes to detach fibroblasts. A suspension containing TE and fibroblasts was collected from the wells and centrifuged at 5000 rpm for 5 minutes. The pellet was suspended in FD+10% FBS and seeded into 75 cm ² flasks. Keratinocytes remaining- on the culture plate were washed with DPBS and supplemented with Epilife for further expansion.

Preparation of dermal fibroblast sera (DFS) As shown in route (10) of Figure 1, human dermal fibroblasts were cultured in FD+10% FBS medium. After reaching 100% confluence, the waste medium was discarded, and cells were washed twice with PBS to remove excess medium. Serum-free keratinocyte-specific medium, i.e. Epilife, supplemented with stimulatory factors (denoted here as KM) were then added to the fibroblast culture. Cells were incubated at 37°C in a 5% C0 ₂ atmosphere for 3 days before collecting the waste medium (DFS), denoted as DFS-KM.

Characterisation of the DFS-KM

The concentration of calcium in the DFS-KM was evaluated using Cobas C 501. There is approximately 0.06 mmol/L of calcium in DFS-KM prepared according to the foregoing description.

Evaluation of the use of the DFS in an epithelial cell culture As shown in route (20) of Figure 1 , the DFS was supplemented to epithelial culture (in KM) to evaluate in vitro expansion of cell for attachment, proliferation, and migration. Such result can be seen in Figure 2(c), and Figure 3 to 6. The cells were observed using time- lapse imaging Nikon A1R-A1 CLSM and analysed via image analysis software NIS Elements AR 3.1 (Nikon).

As a comparison with Figure 2(c), epithelial cells were also grown in medium without DFS, denoted as a keratinocyte-specific basal medium (KM), and a keratinocyte-specific basal medium supplemented with stimulatory factors (KMS). The growth of cells in these cultures can be seen in Figure 2 (a) and (b), respectively. There is significant growth of epithelial cells in cultures represented by Figure 2(b) and 2(c). The result indicates that epithelial cells grow in the presence of stimulatory factors and the fibroblast secreted proteins in DFS.

Figure 3 to 6 show the differences in culturing epithelial cells with DFS-KM and with KMS. Apparently, the concentration of DFS affects the growth of cells. The result indicates that the wound healing effect is mainly due to the presence of fibroblast-secreted proteins from the DFS.

Scratch wound assay was performed to determine the rate of healing to re-populate the scratch area. It was conducted by scratching the confluent monolayer cells with 10 μΐ pipette tip to create a defect. After washing with DPBS, the cells were supplemented with DFS. The images were captured at every 20 minutes for 48 hours using time-lapse imaging system. The wound area was analysed using the NIS-Element AR 3.1 software. As in Figure 6, the rate of healing was increased by supplementation of DFS.

Further, components present in the DFS are listed in Table 1 based on results of liquid chromatography mass spectrometry (LCMS).

Table 1 : Vimentin

Desmin

Peripherin

Alpha-internexin

Serum albumin

Annexin

Putative annexin A2-like protein

Fibronectin

Alpha-actinin-1

Alpha-actinin-4

Alpha-actinin-2

Alpha-actinin-3

Spectrin beta chain, non-erythrocytic 4

Annexin A5

Alpha-enolase

Beta-enolase

Gamma-enolase

Thrombospondin- 1

Pentraxin-related protein PTX3

Actin, cytoplasmic 1

Actin, alpha cardiac muscle 1

POTE ankyrin domain family member E

Putative beta-actin-like protein 3

POTE ankyrin domain family member I

Actin, aortic smooth muscle

Beta-actin-like protein 2

Filamin-A

78 kDa glucose-regulated protein

Heat shock cognate 71 kDa protein

Heat shock 70 kDa protein 1 -like

Heat shock 70 kDa protein 6

Putative heat shock 70 kDa protein 7

Prelamin-A/C

Tropomyosin alpha-4 chain

Triosephosphate isomerase

Annexin A 1

L-lactate dehydrogenase A chain

Collagen alpha- 1 (VI) chain

Galectin-1

Profilin-1

Thioredoxin

14-3-3 protein epsilon 14-3-3 protein zeta/delta

14-3-3 protein gamma

14-3-3 protein beta/alpha

14-3-3 protein theta

14-3-3 protein eta

14-3-3 protein sigma

Lactotransferrin

Transgelin

Heat shock protein HSP 90-alpha

Heat shock protein HSP 90-beta

Heat shock protein 75 kDa, mitochondrial

Endoplasmin

Putative heat shock protein HSP 90-alpha A2

Putative heat shock protein HSP 90-alpha A4

Probable Xaa-Pro aminopeptidase 3

Putative heat shock protein HSP 90-alpha A5

Hemoglobin subunit alpha

Cofilin-1

Cofilin-2

Transgelin-2

Phosphatidylethanolamine-binding protein 1

Decorin

Ubiquitin carboxyl-terminal hydrolase isozyme LI

Proteasome subunit alpha type-6

Vinculin

Glyceraldehyde-3 -phosphate dehydrogenase

Pyruvate kinase PKM

Rab GDP dissociation inhibitor beta

Rab GDP dissociation inhibitor alpha

Protein disulfide-isomerase

Alpha-2-macroglobul in

Pregnancy zone protein

Vasopressin-neurophysin 2-copeptin

Phosphoglycerate kinase 1

Phosphoglycerate kinase 2

Inter-alpha-trypsin inhibitor heavy chain H2

Serotransferrin

Protein deglycase DJ-1

Malate dehydrogenase, cytoplasmic

Tubulin alpha- IB chain

Tubulin alpha- 1 A chain

Tubulin alpha-4A chain Tubulin alpha-3C/D chain

Peroxiredoxin-2

Peroxiredoxin-1

Transketolase

Chromogranin-A

Glucose-6-phosphate isomerase

Prosaposin

Gelsolin

Glutathione S-transferase Mu 2

Fibulin-1

Protein disulfide-isomerase A3

Fructose-bisphosphate aldolase C

Collagen alpha-l(I) chain

Protein S100-A 11

Macrophage migration inhibitory factor

Stathmin

Fructose-bisphosphate aldolase A

Nucleobindin-1

Calmodulin

UTP~glucose-l -phosphate uridylyltransferase

Platelet-activating factor acetylhydrolase IB subunit beta

Caldesmon

Small ubiquitin-related modifier 2

Hemoglobin subunit gamma- 1

Hemoglobin subunit epsilon

Plectin

Moesin

Ezrin

Interstitial collagenase

Annexin A6

Adenosylhomocysteinase 4

Ferritin light chain

Glutathione S-transferase P

Plasminogen activator inhibitor 1

Adenylyl cyclase-associated protein 1

Pigment epithelium-derived factor

Transaldolase

Calumenin

Peptidyl-prolyl cis-trans isomerase A

Hemopexin

Proteasome subunit alpha type-2

Complement C3 Peptidyl-prolyl cis-trans isomerase FKBPIA

Proteasome subunit beta type-3

Elongation factor 2

Ubiquitin-conjugating enzyme E2 variant 1

Macrophage-capping protein

Alpha-fetoprotein

Alpha-2-antiplasmin

Serpin HI

Collagen alpha- 1 (XII) chain

Protein SI 00-A6 ^■

Peroxiredoxin-4

Parathymosin

Nucleoside diphosphate kinase B

Ig gamma- 1 chain C region

Ig gamma-3 chain C region

Thioredoxin domain-containing protein 5

Antithrombin-III

Superoxide dismutase [Cu-Zn]

Nucleolin

Alpha-2-HS-glycoprotein

Protein disulfide-isomerase A6

Plastin-1

Phosphoserine aminotransferase

Secretogranin-2

Metalloproteinase inhibitor 1

Thymosin beta-4

Transitional endoplasmic reticulum ATPase

Elongation factor 1 -gamma

Ribonuclease inhibitor

Rho GDP-dissociation inhibitor 1

Calreticulin-3

ATP synthase subunit beta, mitochondrial

F-actin-capping protein subunit alpha- 1

Proteasome subunit beta type-2

F-actin-capping protein subunit beta

Proteasome subunit alpha type-7

Myosin light chain 6B

Transforming growth factor-beta-induced protein ig-h3

Tubulin beta chain

Endoplasmic reticulum resident protein 29

Secretogranin-3

The present invention may be embodied in other specific forms without departing from its essential characteristics. The described embodiments are to be considered in all aspects only as illustrative and not restrictive. The scope of the invention is, therefore indicated by the appended claims rather than by the foregoing description. All changes, which come within the meaning and range of equivalency of the claims, are to be embraced within their scope.