Therapeutic serum obtained from co-cultured cells

Abstract

A therapeutic serum suitable for inclusion in a cosmetic preparation may be produced by stressing a co-culture including proliferative cells. The co-culture of cells may be obtained by first establishing a monolayer of cells on a surface. After a monolayer of first culture is established, a second culture comprising more resilient and/or aggressive cells may be over-seeded on the monolayer and established. Additional cultures comprising increasingly dominant cells may then be over-seeded and established until a monolayer having the desired population of cells is obtained. The monolayer is then stressed to obtain a serum by conditioning a collection medium. The obtained serum may be combined with a suitable cosmetic base to provide a cosmetic preparation.

Claims

1. A method of producing a serum, comprising the steps of: a) seeding a first surface with a first culture of cells; b) providing the first culture with a first growth medium comprising nutrients and at least one growth factor; c) growing the first culture to a monolayer of less than 100% confluence; d) over-seeding a second culture onto the first surface and the monolayer made in step (c), said second culture comprising cells more dominant than those in the first culture; e) growing the first and second cultures to less than 100% confluence in the presence of the first growth medium; f) after growing the first and second cultures to less than 100% confluence, replacing the first growth medium with a collection medium, said collection medium lacking at least one of the growth factors of the first growth medium; g) maintaining the first and second cultures for a period of time in the presence of the collection medium to produce a conditioned medium; and h) collecting at least a portion of the conditioned medium after the period of time, wherein said conditioned medium is a therapeutic serum.

2. The method of claim 1, further comprising growing the second culture on a second surface to less than 100% confluence before over-seeding onto the first surface.

3. The method of claim 1, wherein the second culture is grown in the presence of a second growth medium before over-seeding onto the first surface, said second growth medium comprising nutrients and at least one growth factor.

4. The method of claim 3, wherein the nutrients of the second growth media are provided by a basal medium.

5. The method of claim 3, wherein the growth factors of the second growth medium comprise at least one selected from the group consisting of L-glutamine, hydrocortisone hemisuccinate, lineolic acid, lecithin, human serum albumin, basic fibroblast growth factor, epidermal growth factor, transforming growth factor, insulin, and vitamin C.

6. The method of claim 1, wherein the nutrients of the first growth media are provided by a basal medium.

7. The method of claim 1, wherein the growth factors of the first growth medium comprise at least one selected from the group consisting of bovine pituitary extract, L-glutamine, hydrocortisone hemisuccinate, transforming growth factor, insulin, epinephrine, and ApoTransferrin.

8. The method of claim 1, wherein the collection medium comprises the nutrients of the first growth medium.

9. The method of claim 1, further comprising: after collecting at least a portion of the conditioned medium, replacing the conditioned medium with the first growth medium; and culturing the first culture and second culture in the first growth medium for a recovery period of time.

10. The method of claim 1, further comprising: before over-seeding the second culture onto the first surface, growing the first culture to a monolayer of approximately 80 to 90% confluence; and creating voids in the monolayer of the first culture.

Description

DETAILED DESCRIPTION

(1) A method for obtaining a therapeutic serum suitable for inclusion in cosmetic preparation now will be described more fully with reference to specific examples. The serum, however, may be obtained in different manners, and thus should not be construed as limited to the specific examples provided. Accordingly, the serum may be obtained by a different ordering and/or sequence of the various steps and/or procedures detailed in the provided examples. For example, two or more steps may be performed concurrently or with partial concurrence. Also, some steps that are performed as discrete steps in the following examples may be combined, and steps being performed as a combined step may be separated into discrete steps, the sequence of certain steps may be reversed or otherwise varied, and the nature or number of discrete steps may be altered or varied. Accordingly, the provided examples are not intended to exclude any of such means of obtaining a therapeutic serum suitable for use in a cosmetic preparation.

(2) Likewise, different reagents, techniques, materials and/or equipment other than those specifically mentioned may be utilized to provide the therapeutic serum.

(3) A therapeutic serum suitable for inclusion in a cosmetic preparation may be produced by stressing a co-culture including proliferative cells. The co-culture of cells may be obtained by first establishing a monolayer of cells on a surface. After a monolayer of a first culture is established, a second culture comprising more resilient and/or aggressive cells may be seeded on the monolayer and established. Additional cultures comprising increasingly dominant cells may then be seeded and established until a monolayer the having the desired cellular composition is obtained. The monolayer is then stressed to obtain a serum by conditioning a collection medium. The obtained serum may be combined with a suitable cosmetic base to provide a cosmetic preparation.

(4) For instance, a serum comprising Human Neonatal Fibroblast/Keratinocyte Conditioned Media may be obtained from a proliferative monolayer comprising a co-culture of keratinocytes and fibroblasts. The co-cultured monolayer may be established by first partially submerging a vial of frozen keratinocytes (obtained from LifeLine Cell Technologies) in a 37 C. water bath, without submerging the top of the vial. The vial is allowed to thaw in the water bath until a small piece of ice remains. The vial is then removed and sprayed with an ethanol solution. In a hood, keratinocytes are seeded from the vial at 2,500 to 5,000 cells per cm.sup.2 onto a culture treated surface. The surface should be provided with an appropriate volume of a suitable growth media, such as a media including Basal DermaLife Media (LifeLine Cell Technologies) and growth factors comprising bovine pituitary extract, L-glutamine, hydrocortisone hemisuccinate, transforming growth factor, insulin, epinephrine and/or ApoTransferrin. The seeded surface is then placed in an incubator and grown at 37 C. in the presence of humidified air comprising 5% CO.sub.2. As to remove any residue DMSO and/or other solvents that may be present in the cryogenic solution, the growth media may be changed every 24 to 48 hours following initiation of the monoculture. After which time, the growth media may be changed every 48 to 72 hours.

(5) Other means of obtaining the initial keratinocytes may also be employed. For instance, keratinocytes may be isolated from neonatal foreskin retrieved from circumcision using the techniques detailed in co-pending U.S. application Ser. No. 14/597,796, filed Jan. 15, 2015, the teachings of which are hereby incorporated by reference in their entirety.

(6) The keratinocytes are allowed to grow in the growth media until 80-90% confluence is achieved. Voids are then created within the established monolayer by removing the growth media and washing twice with an appropriate volume of a buffer solution, such as phosphate buffer solution without calcium or magnesium. After washing with buffer solution, a sufficient volume of an enzymatic cell detachment solution to promote detachment of the cells from the surface is added. For instance, detachment of the cells may be promoted by adding 1 ml of HyQTase Solution, manufactured by HyClone Laboratories, Inc, per 25 cm.sup.2 of growth area. The enzymatically treated monolayer may then be incubated at 37 C. until the keratinocytes start balling. The surface is then tilted to collect the enzyme solution with a pipette. The collected solution is sprayed at focused points onto the monolayer to create voids in about 50% of the monolayer. The enzyme solution and detached keratinocytes are then removed. The remaining monolayer is provided with a sufficient volume of the growth media and returned to the incubator. For example, an amount of media providing 10 ml of media per 55 cm.sup.2 of growth area may be sufficient.

(7) Simultaneously, a monolayer of fibroblasts is cultured on a second surface by submerging a vial of frozen fibroblasts (obtained from LifeLine Cell Technologies) in a 37 C. water bath, without submerging the top of the vial. The vial is allowed to thaw in the water bath until a small piece of ice remains. The vial is then removed and sprayed with an ethanol solution. In a hood, fibroblasts are seeded from the vial at 2,500 to 5,000 cells per cm.sup.2 on to a culture treated surface. The surface should be provided with an appropriate volume of a suitable growth media, such as a media including Basal DermaLife Media (LifeLine Cell Technologies) and growth factors comprising L-glutamine, hydrocortisone hemisuccinate, lineolic acid, licithin, human serum albumin, basic fibroblasts growth factor, epidermal growth factor, transforming growth factor, insulin and/or vitamin C. The seeded surface is then placed in an incubator and grown at 37 C. in the presence of humidified air comprising 5% CO.sub.2. As to remove any residue DMSO and/or other solvents that may be present in the cryogenic solution, the growth media may be changed every 24 to 48 hours following initiation of the monoculture. After which time, the growth media may be changed every 48 to 72 hours.

(8) Other means of obtaining the initial fibroblasts may also be employed. For instance, fibroblasts may be isolated from neonatal foreskin retrieved from circumcision using the techniques detailed in co-pending U.S. application Ser. No. 14/597,796.

(9) When the fibroblast monolayer reaches approximately 80 to 90% confluence, the surface is transferred to a hood and the growth media removed. The fibroblasts monolayer is then washed with a buffer solution, such as phosphate buffer solution without calcium or magnesium. A sufficient volume of a cell detachment solution to promote detachment of the fibroblasts from the second surface is then added. For instance, detachment of the fibroblasts may be promoted by adding 1 ml of Accutase Cell Detachment Solution, manufactured by Innovative Cell Technologies, Inc, per 25 cm.sup.2 of growth area. The fibroblast cells are then incubated in the cell detachment solution at 37 C. until all the cells have detached. A homogenous suspension of cells is then obtained by mixing and approximately 250 l of the fibroblasts suspension is over-seeded onto the keratinocyte monolayer. The over-seeded keratinocyte monolayer is then returned to the incubator. The co-culture is then grown in the keratinocyte growth media until 80 to 95% confluence is achieved.

(10) A monolayer of co-cultured cells may also be achieved by culturing keratinocytes in the keratinocyte growth media until approximately 50% confluence is achieved. The keratinocyte monolayer may then be over-seeded with the cultured fibroblasts suspension. For example, a co-culture in a T175 cm.sup.2 flask would be overlayed with 1.5 ml of fibroblast suspension generated from a confluent T75 cm.sup.2 flask of fibroblasts dissociated using 3 ml of Accutase. The over-seeded keratinocyte monolayer may then be cultured in the incubator until approximately 80 to 95% confluence is achieved.

(11) The co-culture of cells may be stressed to provide a therapeutic serum suitable for use in a cosmetic preparation. Stressing the co-culture may be achieved by selectively removing nutrients, growth factors and/or other favorable conditions. The stress need not be severe. Accordingly, sufficient stress may be induced by removing all or a portion of the growth factors while maintaining nutrient levels. Growth factors may be removed by extracting the keratinocyte growth media from the surface and rinsing the co-culture monolayer twice with a sufficient volume of a buffer solution, such as phosphate buffer solution lacking calcium and magnesium. As to ensure all growth factors are removed, the co-culture may be incubated for a period of time in a collection medium that is added to the surface and then discarded prior to serum collection. For instance, growth factors may be removed prior to serum collection by adding approximately 5.0 ml of a collection medium per 55 cm.sup.2 of growth area and incubating for approximately six hours.

(12) The collection medium may comprise a minimum essential medium with Earl's salt and have the nutrients of the keratinocyte growth medium.

(13) After removal of the growth factors, a sufficient volume of fresh collection medium is added, and the surface returned to the incubator for a sufficient period of time to produce a conditioned medium form the collection medium. For example, incubating the co-culture in approximately 10.0 ml of fresh collection media per 55 cm.sup.2 of growth area for approximately 48 hours may be sufficient to produce a conditioned media from the collection media. After incubating for a sufficient period of time, approximately 50% of the collection media is removed and replaced with an approximately equal amount of fresh collection media. The co-culture is then incubated for approximately 48 hours to produce more conditioned media. After which time, all of the conditioned media is removed.

(14) The co-culture is then allowed to recover by removing the stress and incubating for a period of time. For instance, incubating in the presence of approximately 10.0 ml per 55 cm.sup.2 of growth area of the keratinocyte growth media for approximately 24 to 72 hours may provide sufficient recovery. During recovery, the co-culture may be refreshed by seeding fresh cells of one or more of the cultures onto the monolayer.

(15) After recovering, serum collection is repeated.

(16) The process of the serum collection and recovery may be repeated until the co-cultures no longer produce serum of the desired quality. The quality of serum may begin to degrade when one or more of the cultures used to initially establish the co-culture reach 80% of their life expectancy as defined by the maximum number of population doublings.

(17) The conditioned medium collected may be filtered using a suitable filter, such as a 0.45 m Millipore filter. The serum collected from filtering the conditioned medium may be tested for sterility, virology and/or stability factors.

(18) The therapeutic serum collected may be combined with a suitable cosmetic base as to provide therapeutic cosmetic preparation useful as a recovery cream, moisturizer, neck cream, eye cream and/or facial cream. After combining the therapeutic serum to the base, antioxidants, and/or fragrances may be added to the cosmetic preparation. The cosmetic preparation may comprise approximately 58 to 77 percent by mass of base, approximately 2 to 5 percent by mass of a moisturizer, approximately 9 to 33 percent by mass of the serum, 3 to 14 percent by mass of antioxidants, and approximately 0 to 0.01 percent by mass of fragrance. Other amounts and/or ingredients collectively providing a cosmetically suitable preparation may be utilized in combination with the serum.

(19) In an exemplary embodiment, a facial cream cosmetic preparation may be prepared by adding 2.35 mass percent super low molecular weight hyaluronic acid added to 58.92 mass percent base. The base and hyaluronic acid combination may then be slowly mixed until homogenous. After which, 29.45 mass percent serum may be added to the base and hyaluronic acid mixture and slowly mixed until a homogenous mixture is obtained. Then 5.89 mass percent vitamin C may be slowly mixed in to provide a homogenous mixture. After providing a homogenous mixture including vitamin C, 2.94 mass percent vitamin E may be slowly mixed in to provide a homogenous mixture. Then 0.44 mass percent vitamin A may be mixed in to provide a final homogenous vitamin-base-moisturizer-serum composition. To this composition, 0.01 mass percent fragrance may be mixed in to provide a final homogenous cosmetic preparation.

(20) In an exemplary embodiment, an eye cream cosmetic preparation may be prepared by combining 61.83 mass percent base with 30.90 mass percent serum and slowly mixing until a homogenous mixture of base and serum is obtained. To this mixture, 3.707 mass percent super low molecular weight hyaluronic acid may be slowly mixed in to provide a homogenous mixture. Then, 3.09 mass percent vitamin E may be slowly mixed in to provide another homogenous mixture. To this mixture, 0.463 mass percent vitamin A may be slowly mixed in to provide a homogenous vitamin-base-moisturizer-serum composition. To this composition, 0.01 mass percent fragrance may be slowly mixed in to provide a final homogenous cosmetic preparation.

(21) In an exemplary embodiment, a neck cream cosmetic preparation may be prepared by combining 67.243 mass percent base with 16.810 mass percent serum, and mixing slowly to provide a homogenous mixture of base and serum. To this mixture 4.203 mass percent super low molecular weight hyaluronic acid may be slowly mixed in to provide a homogenous mixture. Then, 7.985 mass percent vitamin C may be slowly mixed in to provide a homogenous mixture. To this homogenous mixture, 3.329 mass percent vitamin E may be slowly mixed in to provide another homogenous mixture. After which, 0.42 mass percent vitamin A may be slowly mixed in to provide a homogenous vitamin-base-moisturizer-serum composition. To this composition, 0.01 mass percent fragrance may be mixed in to provide a final homogenous cosmetic preparation.

(22) In an exemplary embodiment, a moisturizer cream cosmetic preparation may be prepared by combining 76.100 mass percent base with 9.605 mass percent serum and mixing slowly until a homogenous base-serum mixture is obtained. To this mixture, 3.42 mass percent super low molecular weight hyaluronic acid may be slowly mixed to provide another homogenous mixture. To this mixture, 6.60 mass percent vitamin C may be slowly mixed in to provide a homogenous mixture. After which, 3.805 mass percent vitamin E may be slowly mixed in to provide a homogenous mixture. Then, 0.46 mass percent vitamin A may be slowly mixed in to provide a homogenous vitamin-base-serum-moisturizer composition. To this composition, 0.01 mass percent fragrance may be mixed in to provide a cosmetic preparation.

(23) In an exemplary embodiment, a recovery cream cosmetic preparation may be prepared by combining 66.67 mass percent base with 33.33 mass percent serum and mixing slowly until homogenous.

(24) While the present invention has been described herein with respect to the exemplary embodiments, it will become apparent to one of ordinary skill in the art that many modifications, improvements and sub-combinations of the various embodiments, adaptations and variations can be made to the invention without departing from the spirit and scope thereof.