NOVEL APPLICATIONS AND FORMULATIONS OF OPTIMIZED, MODIFIED HUMAN EMBRYONIC FERTILITY CULTURE MEDIA WITH BIGUANIDES AND/OR FUNCTIONAL EQUIVALENTS

Abstract

A dermatological preparation comprises an optimized, modified human embryonic fertility culture media with biguanides and/or functional equivalents and the use of said preparation for skin care, hair care, or body care, or dental procedures, or for regenerative medicine such as promoting wound or bone healing. The preparation is formulated to target the hypoxic microenvironment niches of the skin tissue in combination with optimal concentration of nutrients, ions and minerals; to stimulate existing stem cells to trigger the capacity of stem cells to divide and renew and differentiate into specialized cells; and to stimulate molecular and physiological processes, e.g., autophagy, to replenish the substrate pool through the recycling of organelles and the recycling of old damaged proteins and countering free-radical damage to promote anti-aging.

Claims

1. A dermatological preparation comprising optimized, modified human embryonic fertility medium, said medium further comprising biguanide, wherein the preparation is formulated for application for skin care, hair care, body care, dental procedures, anti-aging, and regenerative medicine such as wound or bone healing.

2. The dermatological preparation of claim 1 wherein the optimized, modified human embryonic fertility medium is simplex optimized, modified human embryonic fertility KSOMMaa medium.

3. The dermatological preparation of claim 1 wherein the optimized, modified human embryonic fertility medium is preimplantation fertility culture media.

4. The dermatological preparation of claim 3 wherein the preimplantation fertility culture media is selected from optimized modified Sydney IVF cleavage medium, Sydney IVF blastocyst medium, FERTICULT medium, FERTICULT G3, IVC-ONE medium, IVC-Two medium, Embryo Assist medium, Blast Assist medium, G-1 v5 Plus medium, G-2 v5 Plus medium, ECM medium, Multiblast Medium, Quinn's Advantage cleavage medium, or a one-step Global medium.

5. The dermatological preparation of claim 1 wherein the optimized modified human embryonic fertility medium is Assisted Reproductive Technology media.

6. The dermatological preparation of claim 5 wherein the Assisted Reproductive Technology media is selected from optimized modified ORIGIO Flushing Medium, SynVitro Flush, Global Collect, global total LP w/HEPES, global total w/HEPES w/HAS, Universal IVF Medium, ORIGIO Sequential Fert, Quinn's Advantage Protein Plus Fert Medium, EmbryoGen, or BlastGen.

7. The dermatological preparation of claim 1 wherein the optimized, modified human embryonic fertility medium comprises a combination of the following components in the following concentration ranges in g/L: TABLE-US-00001 NaCl 4.44 11.1 KCl 0.152 0.38 KH2PO4 0.04 0.1 MgSO47H2O 0.04 0.1 Glucose 0.032 0.08 Sodium lactate 0.896 2.24 NaHCO3 1.68 4.2 Sodium pyruvate 0.016 0.04 CaCl22H2O 0.02 0.05 EDTA 0.0032 0.008 Glycyl-glutamine 0.1168 0.292 L-Glutamine 0.008 0.02 L-Alanine 0.0036 0.009 Glycine 0.003 0.0075 L-Arginine 0.05056 0.1264 L-Asparagine 0.006 0.015 L-Aspartic Acid 0.005328 0.01332 L-Cystine 0.009616 0.02404 L-Histidine 0.016768 0.04192 L-Isoleucine 0.020984 0.05246 L-Leucine 0.020992 0.05248 L-Lysine 0.029216 0.07304 L-Methionine 0.005968 0.01492 L-Phenylalanine 0.013216 0.03304 L-Proline 0.004608 0.01152 L-Serine 0.004208 0.01052 L-Tryptophan 0.004088 0.01022 L-Tyrosine 0.014496 0.03624 L-Valine 0.018736 0.04684

8. The dermatological preparation of claim 7 wherein the one or more optimized, modified human embryonic fertility media comprises biguanide in the concentration range 0.001 g/L-10.0 g/L.

9. The dermatological preparation of claim 7 wherein the one or more optimized, modified human embryonic fertility media comprises one or more functional equivalent of biguanide with a final concentration in the range 0.001 g/L-10.0 g/L.

10. The dermatological preparation of claim 9 wherein the functional equivalent of biguanide is of plant origin.

11. The dermatological preparation of claim 9 wherein the functional equivalent of biguanide is a compound which inhibits mTOR activity or exhibits glucose normalizing properties.

12. The dermatological preparation of claim 9 wherein the functional equivalent of biguanide is Epigallocatechin Gallate.

13. The dermatological preparation of claim 1 wherein the preparation comprises glutamine stabilizing compounds.

14. The dermatological preparation of claim 1 wherein the preparation comprises a dipeptide, tripeptide, or polypeptide, or modified peptide.

15. The dermatological preparation of claim 14 wherein the dipeptide is selected from glycyl-glutamine, alanyl-glutamine, or a mixture of glycyl-glutamine and alanyl-glutamine.

16. The dermatological preparation of claim 1 wherein the preparation comprises ascorbic acid.

17. The dermatological preparation of claim 1 wherein the preparation is present as a concentrate.

18. The dermatological preparation of claim 1 wherein the preparation is present as a ready-mixed powder, granules, or concentrate.

19. The dermatological preparation of claim 1 wherein the preparation is present as an oil-in-water emulsion.

20. The dermatological preparation of claim 1 wherein the preparation is present as a water-in-oil emulsion.

21. The dermatological preparation of claim 1 wherein the preparation is present as a water-in-oil-in-water emulsion.

22. The dermatological preparation of claim 1 wherein the preparation is present as a microemulsion.

23. The dermatological preparation of claim 1 wherein the preparation is present as part of mesotherapy in the form of dermal microinjections or dermal infusions.

24. The dermatological preparation of claim 23 wherein the preparation is a mixture comprising short peptides, DNA sequences, RNA sequences, modified peptides, modified DNA sequences, or modified RNA sequences.

25. The dermatological preparation of claim 23 wherein the preparation is a mixture comprising Argireline (Hexapeptide-8).

26. The dermatological preparation of claim 1 wherein the preparation is present as at least one of a shampoo, a conditioner, a hair mask, or a scalp mask.

27. The dermatological preparation of claim 1 wherein the preparation is present as at least one of a pre-soaked bandage, a dressing, a gauze, a cleansing or rinsing solution.

28. The dermatological preparation of claim 1 wherein the preparation is present as at least one of a surgical suture, a staple, a metal plate, or a diffusing bead.

29. The dermatological preparation of claim 1 wherein the preparation is present in a pharmaceutically appropriate carrier, a stable lipid, micelle, a liposome, a nanoparticle, or a receptor tagged delivery system.

30. The dermatological preparation of claim 1 wherein the preparation is modified for use intravenous, intra-arterial, intramuscular, oral, subdermal, intradermal, or per rectum.

31. The dermatological preparation of claim 1 wherein the preparation is modified for topical eye administration for use on the external skin.

32. A method for making the dermatological preparation of claim 1, wherein the process comprises mixing the one or more optimized, modified human embryonic fertility culture media with biguanide or functional equivalent of biguanide.

33. A method for making optimized modified human embryonic fertility culture media for application for skin care, hair care, or body care, or dental procedures, or for anti-aging, regenerative medicine such as wound or bone healing, comprising the steps of: (a) defining the start and target locations; (b) selecting a first media, defining a start simplex thereof; (c) identifying the composition of said first media; (d) dissecting said start simplex into essential and nonessential components; (e) selecting a target location; (f) identifying the characteristics of the microenvironment niche of said target location; (g) dissecting said target location into essential and nonessential parameters and components in terms of microenvironment metabolic demands, physiology, and microbial abundance; (h) cross-comparing the essential, nonessential components as well as characteristic to mitigate potential negative substrate-substrate interactions between said start simplex and said target location; (i) identifying desired components for optimization thereby defining the new optimized modified media; and, (j) modifying and iterating said optimization until the desired composition is achieved, defining the new optimized modified human embryonic media.

34. A method of claim 33 wherein the first media is KSOM.

35. A method of claim 33 wherein the first media is preimplantation fertility culture media comprising Sydney IVF cleavage medium, Sydney IVF blastocyst medium, FERTICULT medium, FERTICULT G3, IVC-ONE medium, IVC-Two medium, Embryo Assist medium, Blast Assist medium, G-1 v5 Plus medium, G-2 v5 Plus medium, ECM medium, Multiblast Medium, Quinn's Advantage cleavage medium, or a one-step Global medium.

36. A method of claim 33 wherein the first media is Assisted Reproductive Technology media comprising ORIGIO Flushing Medium, SynVitro Flush, Global Collect, global total LP w/HEPES, global total w/HEPES w/HAS, Universal IVF Medium, ORIGIO Sequential Fert, Quinn's Advantage Protein Plus Fert Medium, EmbryoGen, or BlastGen.

37. A method of claim 33 wherein the target location is skin tissue, conjunctiva, or wound or in dental procedures.

38. A method of claim 33 wherein the iteration is performed to optimize conditions and reduce the potential for substrate-substrate interaction.

39. A method of claim 33 wherein the iteration is performed to optimize conditions and target the hypoxic microenvironment niche of the skin, wound or other target tissue.

40. A method of claim 33 wherein the iteration is performed to optimize conditions and stimulate the existing stem cells of S.basale layer and stromal cells in the epidermal/dermal junction.

41. A method of claim 33 wherein the iteration is performed to optimize conditions and create a superior fertility media that comprises mathematically optimized substrate and solute compositions for the microenvironment niches within the skin tissue.

42. A method of claim 33 wherein the iteration is performed to optimized conditions and modulate the metabolic pathways of skin cells to decrease endogenous free-radical DNA damage and promote anti-aging, regeneration, and renewal of the skin.

43. A method of claim 33 wherein the iteration is performed to optimized conditions and modulate the epigenetic profile of the skin to resemble that of younger tissues.

44. A method of claim 33 wherein the iteration is performed to optimized conditions and strengthen the skin as a primary immune barrier and prevent or limit viral, bacterial, fungal or parasitic infection.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Preferred exemplary embodiments of the present invention are illustrated in the accompanying drawings, in which liked reference numerals, if any, represent like parts throughout, and in which:

[0056] FIG. 1 is an advantageous working formulation of the present invention showing suitable composition of optimized, modified human embryonic fertility media.

[0057] FIG. 2 is an advantageous working formulation of the present invention showing optimized composition of modified human embryonic fertility media in combination with biguanide. (LaSecret Formula)

[0058] FIG. 3 is an advantageous working formulation of the invention in a cream form.

[0059] FIG. 4 is an advantageous working formulation of the invention in a gel form.

[0060] FIG. 5 is an advantageous working formulation of the invention in an eye cream form for application on the external skin.

[0061] FIG. 6 is an advantageous working formulation of the invention in a hydrogel form.

[0062] FIG. 7 is an advantageous working formulation of the invention in a spray mist form.

[0063] FIG. 8 is an advantageous working formulation of the invention in a shampoo.

[0064] FIG. 9 is an advantageous working formulation of the invention in a hair conditioner.

[0065] FIG. 10 is an advantageous working formulation of the invention in a face mask.

[0066] FIG. 11 is an advantageous working formulation of the invention in an antiseptic hand spray.

[0067] FIG. 12 is an advantageous working formulation of the invention in a mesotherapy.

[0068] FIG. 13 is a graph representing the effects of the present invention on the metabolic activity/viability of MeWo cells.

[0069] FIG. 14 is a graph representing the effects of the present invention on cell survival ofMeWo cells.

[0070] FIG. 15a and FIG. 15b represent the effects of the present invention on cellular migration and wound healing of MeWo cells

[0071] FIG. 16 is an exemplary, schematic flow chart of the method for making optimized, modified human embryonic fertility media.

DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT

[0072] Simplex optimization as applied to fertility medicine is an algorithm that allows for the determination of optimal substrate concentrations with the endpoint of yielding the highest number of zygote to blastocyst events. It is an efficient method for optimizing culture media by allowing the optimization of many variables simultaneously. The formulation of the present invention described herein contains substrate concentrations that are simplex optimized to support the stem cell phenotype and stem cell metabolism as well as target the hypoxic microenvironment niches of skin tissue.

[0073] As shown in FIG. 1 and FIG. 2, the advantageous working formulation of the invention relates to an optimized, modified human fertility culture media combined with biguanides (LaSecret Formula) for anti-aging and renewal properties. The formulation of the present invention is free from any microbial or other contamination. Use of the invention may be incorporated into a wide range of applications including, but not limited to, the cosmetic industry or tissue regeneration such as wound repair or bone healing. A method for making an optimized, modified human fertility culture media combined with biguanides (LaSecret Formula) is further described herein,

[0074] As shown in FIG. 3, FIG. 4, FIG. 5, FIG. 6, and FIG. 7, the advantageous working formulation of the invention may be prepared as part topical cream, gel, eye cream, hydrogel, or spray mist, respectively. Further, topical cream, sera, and pre-soaked mask of the invention may be used as part of the water phase in the form of an emulsion, suspension, solution, or gel, as shown in, for example, FIG. 3, FIG. 4, FIG. 5, FIG. 6 and FIG. 10. The formulation may further be prepared as a ready-mixed product that may be pre-mixed just prior to application such as in powder, granules, or concentrate syrup form for use, for example, on the epidermis, dermis, or other parts of the human body.

[0075] In yet another embodiment, the present invention may be in the form of a cosmetic product mixed with other substances to achieve the desired properties and the desired result. For instances, substances may be used to modulate texture or moisture or other properties of the product to achieve the desired result. The invention may be modified further to alter pH and/or osmolarity.

[0076] It is further contemplated that formulation of the present invention may be in a concentrate form such that it is incorporated as part of a solution, wherein the final diluted concentration of the substrates will be maintained, and the final concentrations are substantially similar and functionally equivalent to those illustrated in FIG. 1 and FIG. 2.

[0077] A person skilled in the art will appreciate that the formulation of the present invention may be prepared and incorporated into a variety of products. A preferred embodiment of the present invention is the preparation of the invention as part of a water base product. However, the formulation of the invention may be used as part of an emulsion (Oil/Water, Water/Oil, Water/Oil/Water), suspension, solution, or gel of any known defined or undefined medium, as shown in, for example, the advantageous working embodiments described herein.

[0078] Further, an embodiment of the invention may be used as part of mesotherapy in the form of dermal microinjections or infusions. In this regard, the present invention may be used in combination with other substances such as Argireline (Hexapeptide-8) to achieve the desired result as shown in, for example, FIG. 12.

[0079] In yet another embodiment of the invention, a preparation may be applied to the scalp in the form of a shampoo, conditioner, hair mask, or scalp mask as shown in, for example, FIG. 8, FIG. 9, and FIG. 10.

[0080] The formulation of the present invention may also be used to promote wound healing. Wound healing applications may be in the form of pre-soaked bandages, dressings, gauze or cleansing solutions. The formulation of the invention may further be used during surgery and may be incorporatedfor examplein the form of surgical glue to accelerate wound healing.

[0081] In yet another embodiment of the invention, the formulation may further be incorporated into other products including, but not limited to, surgical sutures, staples, or diffusing beads that may be pre-treated with a desired preparation of the present inventionfor example, a dehydrated powder preparation of the invention. In the case of bone fractures, the formulation of the invention may be incorporated into metal plates to accelerate healing and reduce the risk of surgical complications.

[0082] In yet another embodiment of the invention, the formulation of the present invention may be further used in combination with an antibacterial or antiviral or antifungal agent for application as a skin cleanser as shown in the advantageous working formulation of FIG. 11.

[0083] In another embodiment, the formulation of the present invention may be incorporated into a pharmaceutically appropriate carrier or other excipients to achieve the desired bioavailability of the active ingredient. More recently, novel drug delivery systems have changed the landscape of the kinds of molecules that can be transported across cell membranes. For instance, liposomal, nanoparticle, and receptor tagged substances have been shown to increase drug, or substrate delivery and these delivery systems are contemplated in the present invention and may be incorporated to optimize a given formulation of the present invention.

[0084] Another embodiment of the present invention may also be used to prevent hair loss, and/or greying or hair discoloration and/or to stimulate hair growth as shown in the advantageous working formulation of FIG. 8 and FIG. 9.

[0085] Another preferred embodiment of the present invention, a formulation of the invention prepared for topical administration to deliver transdermal nourishment and anti-aging substances to the skin tissue, as shown in, for example, the advantageous working embodiments described herein.

[0086] In another embodiment of the invention, formulation of the invention may be prepared to yield a product suitable for topical eye administration, as shown in the advantageous working formulation of FIG. 5.

[0087] The formulation of the invention may also be prepared in a stable lipid, micelle, liposome, and/or nanoparticle. A person skilled in the art will further appreciate that the formulation of the present invention may be used in combination with substances derived from plants, biological or marine extracts including, but not limited to, peptides, vitamins, emulsifiers, and other minerals or substrates.

[0088] As previously stated, the present invention discloses an advantage over the prior art in at least three respects. For instance, a preferred embodiment of the present invention is specially formulated as disclosed in the advantageous working formulation described herein to stimulate molecular and physiological processes such as, but not limited to, cell migration, cell survival, and cell proliferation and thereby demonstrating anti-aging activity and wound healing properties.

[0089] It is well known in the art that metformin is a biguanide antidiabetic medication that activates the adenosine monophosphate-activate protein kinase (AMPK) pathway and, consequently, inhibits the mammalian target of rapamycin (mTOR) signaling cascade. Metformin further inhibits the respiratory chain complex I in the mitochondria which, in turn, suppresses oxidative phosphorylation. A gross physiological effect is the normalization of blood glucose. High blood-glucose is a known risk factor in the development or cardiovascular disease, stroke and cancer which are the leading causes of death in the developed world. The effects of metformin on cell migration, survival, and proliferation may explain why patients taking metformin live longer or may further explain why old tissues treated with metformin have a similar genetic and proteomic profile as untreated young tissues. It is further known in the art that metformin may regulate metabolism by activating molecular pathways related to low food abundance and hence may decrease cell division. It is further known in the art that decreased cell division due to metformin may be rescued by the presence of Glutamine as an alternative source of fuel instead of glucose. This phenomenon is supported by evidence that glutaminase inhibitors restore the cytostatic phenotype. Since a preferred embodiment of the present invention comprises L-glutamine and Glycyl-Glutamine, cells cultured in an advantageous working formulation of the invention may exhibit decreased cellular metabolism or may show no change in metabolic activity relative to cells cultured in control media (MEM 50 M metformin) depending on the cellular energy needs.

[0090] For example, as shown in FIG. 13, the advantageous working formulation of the present invention (hereinafter also referred to as KSOMaa 50 M metformin) is simplex optimized, modified human embryonic fertility media in combination with metformin to normalize blood glucose levels to modulate the metabolic process and, thereby, control the aging process. To demonstrate the effects of the present invention on metabolic viability of cells, an Alamar Blue Assay was performed using a protocol known to those skilled in the relevant art to measure the metabolic viability of MeWo cells cultured in Potassium Simplex Optimized Medium supplemented with essential and non-essential amino acids (KSOMaa) with 50 M metformin or in KSOMaa alone, or cultured in control media (MEM) with and without 50 M metformin. Those skilled in the art would appreciate that MeWo is a human derived cell line that exhibits a fibroblast morphology with a robust adhesion property that makes this cell line suitable for in vitro experiments.

[0091] As shown in FIG.13, the metabolic activity/viability of MeWo cells was decreased when cultured in KSOMaa 50 M metformin relative to cells cultured in control media (MEM 50 M metformin). Specifically, under starvation conditions (1% FBS), the metabolic activity of MeWo cells cultured in control media (MEM 50 uM metformin) is 10% (**) higher relative to cells cultured in KSOMaa 50 M metformin. Under high serum conditions (10% FBS), the metabolic activity in cells cultured in MEM 50 M metformin is 32% (***) higher relative to cells cultured in KSOMaa 50 M metformin. The decreased metabolic activity of MeWo cells cultured in KSOMaa relative to cells cultured in control media (MEM) demonstrates the anti-aging effects of simplex optimized, modified human embryonic fertility culture media of the present invention since decreased metabolism is correlated with decreased intrinsic aging.

[0092] The results shown in FIG. 13 is further explained by the novel composition of optimized, modified human embryonic fertility culture media of the present invention which is specially formulated to support the transition of zygote to blastocyst prior to host reimplantation thereby increasing the probability of implantation and ultimately the development of a viable human embryo. In contrast, the control media (MEM), including other known and common tissue culture media, lack the novel composition of the optimized, modified human embryonic fertility culture medium of the present invention. Specifically, the control media (MEM) is formulated to support the amplification of neoplastic cells such as, for example, HeLa cells, HEK 293 cells, CHO cells, or other neoplastic cells in culture in a petri dish rather than to support the development of an viable embryo in the human body. The applicant, therefore, contends that the novel composition of optimized, modified human embryonic fertility media of the present invention is far superior to common tissue culture media for application in skin care, hair care, body care, dental procedures, and for anti-aging and regenerative medicine such as promoting wound or bone healing.

[0093] In addition to its effects on cell metabolism, the advantageous working formulation of the present invention also regulates cell proliferation and decrease cell death relative to the effects of control media. The applicant contends that cells cultured in the advantageous working formulation of the present invention will exhibit superior cell or protein content. Greater cell number or protein content may result from either an increase in mitotic rate, or a decrease in cell death with a steady mitotic rate, or some degree of both. As disclosed in the experimental results of FIG. 13, since MeWo cells cultured in the advantageous working formulation of the present invention (KSOMaa 50 M metformin) showed a decreased metabolism, it stands to reason that an increase in cell number or protein content would not result from an increased mitosis rate. To further test the beneficial properties of a preferred embodiment of the present invention, a Sultorhodamine B (SRB) study was conducted to determine cell survival and cytotoxicity as a measure of absolute protein content that is related to cell number.

[0094] As shown in FIG. 14, and consistent with the anti-aging properties of the present invention, the protein content or cell number of MeWo cells was significantly higher when cells were cultured in an advantageous working formulation of the present invention (KSOMaa 50 M metformin) as compared to cells cultured in control media (MEM 50 M metformin). Specifically, MeWo cells cultured in the working formulation of the present invention showed an approximately 30%(***) increase in cell number relative to cells cultured in control media MEM +/50 M metformin, thereby demonstrating the beneficial effects of the preferred embodiment of the present invention on cell proliferation and its anti-aging properties. As described herein, the SRB study is an indicator of protein abundance from live cells and thus correlates with relative cell number between experimental conditions. Since simplex optimized, modified human embryonic fertility culture media with 50 M metformin of the present invention does not increase cell metabolism, the effects shown in FIG. 14 suggest that cells cultured in the working formulation of the present invention live longer and are better equipped to proliferate to achieve a net effect of increased protein content. In the context of regenerative medicine, the increase in overall cell number and protein content is thought to support de novo collagen synthesis with limited loss. Accordingly, the applicant contends that the simplex optimized, modified human embryonic fertility culture media in combination with biguanide of the present invention is superior to the existing art.

[0095] To further demonstrate an advantage of the present invention over the prior art, a wound healing study was conducted using the advantageous working formulation of the present invention and MeWo cells. As shown in FIG. 15a and FIG. 15b, and consistent with the regenerative properties of the present invention, a functional assessment of molecular and physiological processes of MeWo cells cultured in KSOMaa 50 M metformin or cultured in KSOMaa alone demonstrates a higher wound closure when compared to cells cultured in control media (MEM +/50 M metformin). Microscopy images as shown in FIG. 15a depict the wound closure over time as measured at t=0, 24, 48, and 72 hours. As shown in FIG. 15b, the wound closure is represented in a graph quantified as the area of the wound (gap) at each time reference plotted as a ratio of said area and the initial area at t=0.

[0096] Accordingly, the data demonstrates that cells cultured in an advantageous working formulation of the present invention are induced in a time-dependent manner to migrate and close the gap to promote wound healing. Consistent with this effect and, as shown in FIG. 15a, it may be qualitatively observed that cells cultured in KSOMaa demonstrate significantly increased podocyte extension which is consistent with cell migration into the direction of wound opening, i.e., a closing of the gap. This qualitative observation suggest that the beneficial effects of the present invention is derived from the novel composition found in simplex optimized modified human embryonic fertility media of the present invention. Therefore, the applicant contends that an advantage of the present invention over the prior art includes the stimulation of essential molecular and physiological processes leading to cell migration, cell survival, and cell proliferation and, thereby, demonstrating anti-aging activity and wound healing.

[0097] In yet another advantage over the prior art, a preferred embodiment of the present invention is specially formulated as disclosed in the advantageous working formulation described herein to target the hypoxic microenvironment niches of the skin tissue in combination with optimal concentration of nutrients, ions, and minerals. A monolayer of cell culture is a useful in vitro scientific model to study basic cell behavior and molecular processes. However, a monolayer of cell culture is an unreliable model to study in vivo phenomena such as understanding the dynamic microenvironment present in tissues and organs. The dynamic microenvironment results in part from gradients in oxygen levels, substrates, and other metabolites. For example, normoxic cells directly adjacent to an oxygen source may produce ATP via oxidative phosphorylation and glucose. In contrast, hypoxic cells will produce ATP via glycolysis and the incorporation of L-Glutamine into the Krebs's cycle; consequently, decreasing the microenvironment pH and increasing lactic acid production in hypoxic tissue regions.

[0098] One approach to partly overcome the limitations of the in vitro monolayer culture is to develop a working model of the dynamic microenvironment of multicellular tissue through spheroid formation. Spheroids are spherical clumps of cells grown on a non-adhesive medium such as agar. It is well known in the art that cells located within the core of a spheroid are in a hypoxic microenvironment. Various culture conditions aimed at alleviating or normalizing hypoxia may be measured using a nitroimidazole compound called EF5. Standard assays and protocols known in the art have been developed to stain for EF5 using immunohistochemistry or immunofluorescence. MeWo cells have the capacity to form spheroids when grown under controlled culture conditions in a non-adhesive medium such as agar. The MeWo spheroids may then be stained for EF5 to assess the level of hypoxia. The advantageous working formulation of the present invention, for example, but not limited to, KSOMaa 50 M metformin, is specially formulated to decrease EF5 staining which is indicative of decrease hypoxia in MeWo spheroids.

[0099] In yet another advantage over the prior art, a preferred embodiment of the present invention is specially formulated as disclosed in the advantageous working formulation described herein to stimulate existing stem cells to divide, renew and differentiate into specialized cells. Within the deepest layer of the skin, S basale of the epidermis, actively dividing and differentiating stem cells lead to improved tissue architecture of skin, improved skin morphology, collagen density, and many other markers of healthy skin such as moisture content. In vivo reflective confocal microscopy is a known procedure for assessing the 3-Dimensional structure of human skin including tissue architecture, morphology, collagen density, and moisture content. It is a preferred means for examining the health of human skin due to the observable depth of tissue penetration.

[0100] A preferred embodiment of the present invention is specially formulated to promote healthy skin moisture content as well as long-term improvement of collagen density, tissue architecture, and morphology of the skin. The beneficial effects of the present invention may be observed using in vivo reflective confocal microscopy. As disclosed in FIG. 13, FIG. 14, FIG. 15a, and FIG. 15b, a preferred embodiment of the present invention demonstrates the capacity to stimulate essential molecular and physiological processes leading to cell migration, cell survival, and cell proliferation and, thereby, demonstrating anti-aging activity and wound healing. Consistent with the experimental results disclosed herein, a topical formulation of the present invention is specially formulated to promote healthy human skin.

[0101] To further promote healthy human skin, a preferred embodiment of the present invention is formulated to stimulate the stress-coping mechanisms of skin cells and inhibit cell apoptosis. Aging cells are under stress which, in turn, trigger changes in the expression of essential genes involved in the cellular-stress response such as apoptosis, or autophagy/unfolded protein response. A preferred embodiment of the present invention is formulated to modulate gene expression of essential genes involved in the cellular-stress response, which may be assessed and quantified using reverse transcribed quantitative polymerase chain reaction (RT-qPCR) and SYBR-green. For instance, a preferred embodiment of the present invention is formulated to decrease the gene expression of genes involved in apoptosis such as BCL2 and CASP3, or to decrease the gene expression of genes implicated in autophagy/unfolded protein response such as BIP, CHOP, XBP 1. A full panel of related transcripts may be measured such as BIP, CHOP, XBP1, BCL2, CASP3, GADD45a, IGFBP2, HIF1a, CDKN1A, TP53, GAPDH, and RN18S1.

[0102] In addition, a preferred embodiment of the present invention is formulated to not only modulate gene expression but also to affect the protein expression of essential proteins involved in the cellular-stress response. It is well known in the art that immunofluorescence is a preferred means to assess the intracellular organelles and cellular markers induced as a result of stress or autophagy on a per cell basis. Under stress conditions, cells up-regulate various pathways such as the unfolded protein response (UPR) or autophagy which may lead to intracellular degradation of organelles and changes in lysosomal number (Mannose-6-phosphate-receptor) as well as endosomes (early endosome antigen 1). Cells undergoing autophagy demonstrate changes in punctate fluorescence corresponding to M6PR tagged lysosomes in the cytoplasm. A preferred embodiment of the present invention is formulated to down-regulate and inhibit the various pathways leading to intracellular degradation including decreased lysosomal vacuoles and endosomes. The applicant, therefore, contends that an advantage of the present invention over the prior art includes targeting the hypoxic microenvironment of the skin and stimulating existing stem cells to divide, renew, and differentiate to promote healthy skin.

[0103] In another preferred embodiment of the present invention, a method for making optimized, modified human embryonic fertility media is disclosed as shown in, for example, FIG. 16.

[0104] A method comprising the steps: (a) defining a start and target location; (b) selecting and defining a first media, START simplex, that has been shown to be safe and effective, wherein said first media is KSOM, one-step or two-step preimplantation fertility culture media, or Assisted Reproductive Technology (ART) media. The one-step or two-step preimplantation media comprising Sydney IVF cleavage medium, Sydney IVF blastocyst medium, FERTICULT medium, FERTICULT G3, IVC-ONE, IVC-Two medium, Embryo Assist, Blast Assist, G-1 v5 Plus, G-2 v5 Plus, ECM medium, Multiblast Medium, Quinn's Advantage cleavage, or a one-step Global medium. The ART media comprising ORIGIO Flushing Medium, SynVitro Flush, Global Collect, global total LP w/HEPES, global total w/HEPES w/HAS, Universal IVF Medium, ORIGIO Sequential Fert, Quinn's Advantage Protein Plus Fert Medium, EmbryoGen, or BlastGen; (c) identifying the composition of said first media including osmolarity, pH, nutrients, preservatives, and energy substrates, e.g., glucose and TCA intermediates to thoroughly understand the role that each substrate composition plays including, for example, whether it is to provide a carbon-source, a co-factor, pH regulator, or as an osmotic agent; if necessary, further (d) dissecting the composition into essential and non-essential substances.

[0105] Whether a substance is essential depends on the preparation and application; (e) selecting a TARGET location, e.g., skin tissue; (f) identifying the specific characteristics of the microenvironment niche of the TARGET location which will dictate the final formulation. Key parameters include osmolarity, pH, presence of commensal and non-commensal microorganisms, oxygenation, blood perfusion, presence of immune system cells and other physiological characteristics. The identified target components for optimization in the new media may be oxygenation, fluid-loss, hypo/hyper-thermia, substrates such as essential and non-essential amino acids, lipid profiles, other substrates such as EDTA, Pyruvate, KH.sub.2PO.sub.4, Glucose, cytokines, proteins, peptides, electrolytes and other desirable components.

[0106] Once key characteristics are identified in the target location, they are divided between essential and non-essential substances ((g) dissecting the TARGET location). Again, whether a substance is essential depends on the preparation and application. A comparison between the START and TARGET location essential and non-essential components is accounted for during the first cycle of media modification ((h) Cross Comparing the START and TARGET). The preparation is further optimized to limit and preferably to eliminate any substrate-substrate interactions which may result in one substrate exhibiting countervailing properties of another. These interactions are synonymous to drug-drug interactions in pharmacology where the presence of one drug may alter the therapeutic effects of another. Additional cycles of media modification and optimization may be necessary depending on the START simplex and TARGET location.

[0107] Objective tests, well known to a person skilled in the art, may be performed to assess the effectiveness of the optimized preparation through its formulation into, for example, a topical cream and the effects of said cream maybe measured through in vivo reflexive confocal microscopy to assess the altered refractive index of the epidermis (skin hydration), or during more extended studies, the effects of said cream on collagen may be measured.

[0108] The method of the present invention is iterative (Steps (i) and (j)) and a few cycles of optimization of the START simplex to reflect the needs of the TARGET location may be necessary to produce a desired product, e.g., See FIG. 16.

[0109] It is therefore submitted that the instant invention has been shown and described in what is considered to be the most practical and preferred embodiments. It is recognized, however, that departures may be made within the scope of the invention and that obvious modifications will occur to a person skilled in the art. With respect to the above descriptions then, it is to be realized that the present invention including the optimum steps and the optimum sequence of steps of the invention to include variations in media composition (i.e., substrates) as well as media conditionsdepending on its intended applicationare deemed readily apparent and obvious to one skilled in the art, and all equivalent relationships to those illustrated in the drawings and described in the specifications are intended to be encompassed by the present invention.

[0110] Therefore, the foregoing is considered as illustrative only of the principles of the invention. Further, since numerous modifications and changes will readily occur to those skilled in the art, it is not desired to limit the invention to the exact construction and operation shown and described, and accordingly, all suitable modification and equivalents may be resorted to, falling within the scope of the invention.