TW202421775A - Composition and Method of Treatment for Cell Growth Using Antler Stem Cell-Conditioned Medium - Google Patents

Abstract

A cell growth composition comprising antler stem cell-conditioned medium (ASC-CM) encapsulated in nanoparticle is disclosed. A cell growth composition comprising ASC-CM obtained from antler stem cell cultured in Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum (FBS) and streptomycin is further disclosed. A method of treatment for cell growth comprising administering one of the cell growth compositions is still further disclosed.

Description

Compositions and methods for treating cell growth using velvet antler stem cell-conditioned medium

The present invention relates to a composition and method for treating cell growth using antler stem cell-conditioned medium.

It is known that antler velvet, once shed from its horn, completely regenerates due to a stem cell-based process; specifically, previously completed studies have provided evidence that antler stem cells not only completely generate antler velvet, but also promote the perfect healing of antler skin wounds; therefore, there is a need to utilize these compounds produced by and associated with antler stem cells for application in the human body to promote cell growth and healing.

Therefore, the applicant must develop the invention and file an application.

The purpose of the present invention is to provide a composition and method for treating cell growth using antler stem cell-conditioned medium, wherein the cell growth composition comprises antler stem cell-conditioned medium (ASC-CM) encapsulated in nanoparticles.

The purpose of the present invention is to provide a composition and method for treating cell growth using antler stem cell-conditioned medium, wherein the cell growth composition comprises antler stem cell conditioned medium (ASC-CM) obtained from antler stem cells cultured in Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS) and streptomycin.

The purpose of the present invention is to provide a composition and method for treating cell growth using velvet antler stem cell-conditioned medium, wherein the cell growth composition includes a drug administration step of one of the above-mentioned cell growth compositions.

Figure 1: Schematic diagram of the experimental procedure for cell growth in antler stem cell-conditioned medium (ASC-CM).

Figure 2: A schematic diagram depicting the preparation process of ASC-CM.

Figure 3: Depicting the results of an experiment using ASC-CM to grow MDCK cells (Madden-Darby canine renal epithelial cells).

Figure 4: Depicts the MDCK cell growth results under a microscope.

Figure 5: Depicts the results of an experiment using ASC-CM to grow NIH-3T3 (mouse embryonic fibroblast cell line).

Figure 6: Depicts the NIH-3T3 cell growth results under a microscope.

Figure 7: A schematic diagram illustrating an embodiment of a method for preparing ASC-CM encapsulated in nanoparticles.

Figure 8: List the characteristics of ASC-CM nanoparticles.

The present invention discloses a composition and method for treating cell growth using antler stem cell-conditioned medium, wherein the composition may include the basic elements and limitations of the present invention described herein and any additional or optional ingredients, components or limitations described herein, and the composition is composed of or substantially composed of the above items.

The present invention discloses a composition and method for treating cell growth using antler stem cell-conditioned medium. The singular forms "a", "an" and "the" used in this specification and the attached patent application also include plural references (unless the context clearly stipulates otherwise). For example, the term "a" cell includes plural cells (including mixtures thereof).

The present invention discloses a composition and method for treating cell growth using velvet antler stem cell-conditioned medium. In the context of a quantity value, "approximately" means a maximum deviation of ±20%, preferably ±10%, or more preferably ±5% based on the average deviation of the value; for example, relative to the molar concentration of total lipid/amphiphile, an amount of about 30 mol% anionic lipid means 30 mol% ±6 mol% anionic lipid, preferably 30 mol% ±3 mol%, or more preferably 30 mol% ±1.5 mol%.

The present invention is a composition and method for treating cell growth using velvet antler stem cell-conditioned medium, wherein "lipid" refers to its traditional meaning as a general term, including fat, lipids and alcohol-ether soluble components of protoplasm (which are insoluble in water); lipids are composed of fats, fatty oils, essential oils, waxes, steroids, sterols, phospholipids, carbohydrates, sulfolipids, amine lipids, chromophores and fatty acids, and the term includes both naturally occurring lipids and synthetic lipids; preferred lipids related to the present invention are: steroids and sterols, especially cholesterol, phospholipids, including phospholipids Acyl and phosphatidylcholine and phosphatidylethanolamine, as well as sphingomyelin; fatty acids, when present, may be about 12 to 24 carbon chains in length, with up to 6 double bonds; fatty acids are attached to a backbone chain, which may be derived from glycerol; fatty acids within a lipid may be different (asymmetric), or only one fatty acid chain may be present, such as lysolecithin; mixed formulations may also be present, especially when the non-cationic lipid is obtained from a natural source, such as purified lecithin (phosphatidylcholine) from egg yolk, beef heart, brain or liver, or soy.

The present invention provides a composition and method for treating cell growth using antler stem cell-conditioned medium, wherein the "effective amount" is an amount sufficient to produce a beneficial or desired result; the effective amount can be administered in one or more administrations, uses or dosages.

The present invention discloses a composition and method for treating cell growth using antler stem cell-conditioned medium. The terms "subject", "individual" or "patient" are used interchangeably herein and refer to vertebrates, preferably mammals, and more preferably humans; mammals include (but are not limited to) mice, monkeys, humans, farm animals, sports animals and pets.

The present invention is a composition and method for treating cell growth using antler stem cell-conditioned medium. Antler is the only known mammalian organ that can completely regenerate when it falls off its horn stalk, and it is known that wounds will heal without leaving scars. The following example describes the therapeutic effect of ASC-CM on cell growth, indicating that ASC-CM is composed of multiple compounds that promote human cell growth.

The present invention discloses a composition and method for treating cell growth using antler stem cell-conditioned medium, wherein the composition comprises ASC-CM encapsulated in nanoparticles such as vesicles or lipid nanoparticles (LNPs), which can be administered orally, topically or by injection; in another embodiment, the ASC-CM composition of the present invention comprises lipid micelles encapsulating ASC-CM; in yet another embodiment, the ASC-CM composition of the present invention comprises vesicles encapsulating ASC-CM.

The present invention discloses a composition and method for treating cell growth using antler stem cell-conditioned medium, wherein the composition includes a freeze-dried form of ASC-CM encapsulated in the above-mentioned nanoparticles, and the freeze-dried ASC-CM nanoparticles can be reconstituted for injection; in one embodiment, the size of each nanoparticle is between about 100 nanometers and about 10,000 nanometers, between about 250 nanometers and about 7,500 nanometers, or between about 500 nanometers and about 5,000 nanometers; the composition of the present invention also includes a shaping agent or adjuvant, and possible shaping agent examples include sodium aluminum sulfate or sucrose.

The present invention discloses a composition and method for treating cell growth using velvet antler stem cell-conditioned medium, which can also be combined with one or more pharmaceutically acceptable excipients or adjuvants to prepare the ASC-CM composition of the present invention in the form of a capsule encapsulated with ASC-CM, such as a gel cap, for oral administration. In one embodiment, the cell growth composition of the present invention includes the nanoparticle AS disclosed herein. In any embodiment of ASC-CM, the nanoparticles ASC-CM are encapsulated in a capsule such as a gel cap for oral administration; in another embodiment, the cell growth composition of the present invention includes any embodiment of ASC-CM nanoparticles in the form of a freeze-dried powder disclosed herein, which is encapsulated in a capsule for oral administration. An embodiment of such a preparation will be disclosed in the following embodiments.

The present invention discloses a composition and method for treating cell growth using antler stem cell-conditioned medium. The composition of the present invention can also be formulated for external administration. In one embodiment, the composition includes any form of ASC-CM composition disclosed above and a pharmaceutically acceptable excipient or adjuvant, which is packaged in a manually destructible tube, which can be easily destructed by hand to release ASC-CM for external administration.

The present invention relates to a composition and method for treating cell growth using antler stem cell-conditioned medium. All preferred embodiments discussed for one or more aspects of the present invention also relate to all other aspects; this particularly refers to the amount and type of neutral or anionic lipids, the amount and type of active agents, the amount and type of further active agents used in combination therapy, and the type of disease to be treated.

The present invention discloses a composition and method for treating cell growth using antler stem cell-conditioned medium. The following embodiments are intended to be illustrative only and are not intended to limit the scope of the present invention. Other general and specific configurations will be apparent to those skilled in the art.

First, please refer to Figures 1 to 8 and the following detailed description: The present invention discloses a composition and method for treating cell growth using velvet antler stem cell-conditioned medium, and Examples 1 and 2 use MDCK and NIH-3T3 cells grown using ASC-CM.

Antlers were collected from healthy sika deer, and small antler tip segments of about 10 mm × 5 mm × 5 mm were collected from them; the collected antler tip segments were washed once with 75% alcohol, and then washed twice with phosphate buffered saline (PBS); then the antler tip segments were cut into 1 mm smaller segments in a 10 cm culture dish, and washed again with PBS; then the small slices were centrifuged at a speed of 200xG for 2 minutes, PBS was removed and antler stem cells (ASCs) were isolated; the isolated ASCs were plated at 1×10 ASCs were seeded at a density of ⁵ cells/well in a six-well plate. Then, 1 ml of 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA) was added to ASCs and cultured at 37°C for 1 hour. Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) was then added and ASCs were cultured for 72 hours, during which the growth medium was replaced every 2 to 3 days. The supernatant was then collected as ASC-CM for experiments.

FIG2 depicts the preparation of the Madden-Darby canine renal epithelial cell (MDCK cell) experimental group; 1 ml of ASC-CM containing 10% FBS was mixed with 4 ml of DMEM containing 10% FBS to form 5 ml of growth medium containing 10% FBS, 20% ASC-CM and 70% DMEM; the 5 ml of growth medium was further mixed with 15 ml of DMEM without FBS to form 20 ml of growth medium containing 2.5% FBS, 5% ACM and 92.5% DMEM; in addition, to separate the cells from the supernatant, 200 g of MDCK cells were centrifuged for about 10 minutes; then the cells separated by centrifugation were centrifuged at 3×10 ⁵ cells/ml in DMEM without FBS; then 1 ml of this MDCK cell DMEM culture was placed in each well and diluted with 1 ml of the above 20 ml growth medium to form a 1.25% FBS + 2.5% ASC-CM and 96.25% DMEM solution containing MDCK cells.

Preparation of the MDCK cell control group involved the preparation of a growth medium containing 2.5% FBS and 97.5% DMEM; 1 ml of this growth medium was then mixed with 1 ml of MDCK cells in DMEM to form a growth medium containing 1.25% FBS and 98.75% DMEM containing MDCK cells.

All wells were cultured in a Forma Series II 3 110 temperature-controlled incubator at 37°C 5x _CO2 ; samples were obtained from the wells on days 1, 3 and 5; the MDCK cells were washed twice with PBS, and 0.5 ml of trypsin was added to detach the MDCK cells for MDCK cell counting; the resulting counts are shown in Figure 3, where the 0% curve represents the control group without ASC-CM, and the 20% curve represents the experimental group in which ASC-CM contained 20% of the culture medium.

As shown in Figure 3, after 5 days of culture, the difference in MDCK cell counts between 0% ASC-CM and 20% ASC-CM culture media was greater than 20%; Figure 4 shows the comparison of cell apoptosis in 0% ASC-CM culture media and MDCK cell proliferation in 20% ASC-CM culture media under a microscope; both Figures 3 and 4 support the results that ASC-CM stimulates MDCK cell proliferation.

The same experiment was performed on NIH-3T3 cells, and the results are shown in Figures 5 and 6. As shown in Figure 5, even after 2 days of culture, the difference in MDCK cell counts between 0% ASC-CM and 20% ASC-CM medium was greater than 10%. The difference in cell counts increased to more than 20% on day 3, and nearly 30% on day 6. Figure 6 shows the difference in cell apoptosis in 0% ASC-CM medium and 20% ASC-CM medium under a microscope. Comparison of NIH-3T3 cell proliferation in ASC-CM medium: Figures 5 and 6 Both support the results that ASC-CM stimulates NIH-3T3 cell proliferation; the same experiment can be performed on human retinal pigment epithelial cells (ARPE-19) and rat cardiomyocytes (H9c2) to demonstrate the therapeutic effect on cell growth of different cell types.

Example 3: Preparation of ASC-CM nanoparticles using PLGA particle delivery system:

FIG7 depicts the preparation of ASC-CM nanoparticles using a PLGA particle delivery system; the process begins with the preparation of an aqueous solution containing ASC-CM and an oily solution containing lipids; specifically, the aqueous solution comprises approximately 200 μL of a protein solution containing ASC-CM at a concentration of approximately 14.4 mg/mL in a 0.5% polyvinyl alcohol (PVA) phosphate buffered saline (PBS) solution; the oily solution contains 200 mg of Resomer® RG502H poly(D,L-lactide-co-glycolide) (RG502H PLGA) in 2 mL of dichloromethane (DCM), wherein RG502H PLGA accounts for approximately 10% of the weight volume of the oily solution.

The aqueous phase solution was then mixed with the oil phase solution in a homogenizer (IKA) and stirred at a speed of about 5 for about 1.5 minutes to form an oil-in-water emulsion. The emulsion was then added to about 15 ml of a 5% by weight PVA solution, and the resulting emulsion was again stirred at a speed of about 5 for about 1.5 minutes to form an oil-in-water double emulsion.

In the next step, the oil-in-water double emulsion was subjected to a solvent evaporation process at 4°C for about 2 hours to form a suspension; the suspension was then centrifuged at a rate of about 300xG for about 5 minutes to remove large hard lumps; then about 15.5 ml of the supernatant was centrifuged at a rate of about 21,000xG for about 15 minutes; then the nanoparticle pellets were washed once and centrifuged at a rate of about 500xG for about 3 minutes; about 15.5 ml of The aliquot of the supernatant is centrifuged again at a rate of about 21,000xG for about 15 minutes; the result is then resuspended in about 1.9 ml of particle-free deionized water; the suspension is freeze-dried at about -80°C and stored; or the ASC-CM nanoparticles in about 1.9 ml of particle-free deionized water are mixed with about 10 ml of 25% sodium aluminum sulfate and 75% sucrose, then freeze-dried into a powder form and packaged into capsules for oral administration.

The freeze-dried nanoparticles were then dissolved in PBS buffer at pH 7.2 for characterization using dynamic light scattering (DLS); the results summarized in Figure 8 show a Z-average diameter of approximately 444.9 nm and a PDT (photodynamic therapy) of 0.265.

In summary, this invention does meet the requirements of Article 21 of the Patent Law. We sincerely request the Bureau to review and grant the legal patent right. We are grateful.

Claims (13)

A composition and method for treating cell growth using antler stem cell-conditioned medium, wherein the cell growth composition comprises antler stem cell-conditioned medium (ASC-CM) encapsulated in nanoparticles. As described in Item 1 of the patent application, a composition and method for treating cell growth using antler stem cell-conditioned medium, wherein the nanoparticles of the composition include lipid micelles. As described in Item 1 of the patent application, a composition and method for treating cell growth using antler stem cell-conditioned medium, wherein the nanoparticles of the composition include liposomes. As described in claim 1, a composition and method for treating cell growth using antler stem cell-conditioned medium, wherein the nanoparticles of the composition contain poly (D, L-lactide-co-glycolide) (PLGA). As described in Item 1 of the patent application, a composition and method for treating cell growth using antler stem cell-conditioned medium, wherein the composition is freeze-dried. As described in Item 1 of the patent application, a composition and method for treating cell growth using antler stem cell-conditioned medium, wherein the composition includes sodium aluminum sulfate and sucrose. A composition and method for treating cell growth using antler stem cell-conditioned medium, comprising antler stem cell-conditioned medium (ASC-CM) obtained from antler stem cells cultured in Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS) and streptomycin. As described in Item 7 of the patent application, a composition and method for treating cell growth using antler stem cell-conditioned medium, wherein the composition also contains sodium aluminum sulfate and 75% sucrose. As described in Item 7 of the patent application, a composition and method for treating cell growth using antler stem cell-conditioned medium, wherein the composition further comprises a capsule encapsulating the ASC-CM. A composition and method for treating cell growth using antler stem cell-conditioned medium, including the step of administering any composition as described in item 1 or 7 of the patent application scope. As described in Item 10 of the patent application, a composition and method for treating cell growth using antler stem cell-conditioned medium, wherein the administration step includes administration by injection. As described in Item 10 of the patent application, a composition and method for treating cell growth using antler stem cell-conditioned medium, wherein the administration step includes oral administration. As described in Item 10 of the patent application, a composition and method for treating cell growth using antler stem cell-conditioned medium, wherein the administration step includes external administration.