Methods for preparing human cellular materials, compositions thereof, and their applications

Abstract

A method for producing and applying cellular materials from human cells for non-therapeutic applications is provided. The method comprises the steps of obtaining human cells, cultivating the human cells in a controlled environment, harvesting the cultivated human cells, processing the cultivated human cells to produce cellular materials and applying the cellular materials to a human subject for non-therapeutic applications. Other example embodiments are described herein. In certain embodiments, the method offers an environmentally sustainable solution that provides human cellular materials having improved safety, bioactivity, and batch consistency for human use.

Description

Attorney document no.: 233490- 40180METHODS FOR PREPARING HUMAN CELLULAR MATERIALS, COMPOSITIONS THEREOF, AND THEIR APPLICATIONSTECHNICAL FIELD

[0001] Embodiments discussed herein generally relate to methods, compositions and uses involving cellular materials derived from human cells.BACKGROUND

[0002] Cells contain numerous bioactive materials, including proteins, peptides, amino acids, phospholipids, free fatty acids, glycans, trace minerals, and antioxidants that support life. Cells in different parts of the body vary in composition and can be extracted and used in forms such as whole cells, total lysates, hydrolysates, and targeted proteins. These cellular materials serve as active ingredients in many applications, including dietary supplements, hair care, skin care, cosmetic products, and wound care. Whole cells, such as skin cells and blood cells, have been extensively used for medical treatment. Tissues, such as the placenta, have long been utilized as extracts or supplements, and can also be applied topically. Growth factors, cytokines, and extracellular matrix proteins may stimulate skin tissue repair and regeneration, while fibulins and connective tissue growth factor (CTGF) may accelerate wound healing.

[0003] Traditionally, bioactive materials have been sourced from animal and plant tissues. However, this approach presents several limitations. One significant issue is the reduced efficacy of materials derived from non-human species, which may have suboptimal effects due to differences in composition and proportion compared to their human counterparts. This often necessitates the use of mega-doses or additional purification to compensate for poor efficacy, which can be both costly and resource-intensive.

[0004] Safety and immunogenicity concerns also arise when using products manufactured from non-human origin components. These products may raise consumer concerns regarding safety and allergic reactions, as whole cells and cell lysates from non-human sources contain immunogens and allergens. Additionally, contamination risks are associated with raw materials derived from biological sources. These materials may contain environmental pollutants or pathogens that can inadvertently be incorporated into the final product, posing risks to consumer safety.

[0005] Constant sourcing of raw materials from animals and plants also creates environmental burdens, raises animal welfare concerns, and can disrupt ecosystems andHkl461873.1922222-14296Attorney document no.: 233490-10003 biodiversity. The practices of livestock fanning and plant harvesting can lead to deforestation, increased water consumption, and pollution from animal waste and pesticides. Additionally, the reliance on animal -derived materials often requires animal sacrifice, which raises ethical concerns.

[0006] Another challenge is the batch-to-batch variability inherent in raw materials sourced from animals, fish, and plants. This inconsistency complicates the formulation of reliable products and can affect their efficacy. Processing challenges also emerge, as extensive purification is often required to isolate target molecules from non-human sources. This process leads to high resource consumption and the potential loss of bioactive compounds that could be beneficial.

[0007] Alternative approaches, such as recombinant protein expression and chemical synthesis, have their drawbacks. For instance, recombinant proteins produced in microbial hosts may lack the proper post-translational modifications necessary for full functionality. While mammalian expression systems can produce more complex proteins, they are limited in the number of proteins that can be expressed simultaneously. Moreover, genetically modified organisms used for recombinant expression pose potential environmental risks, particularly if they are accidentally released. Chemical synthesis of active ingredients involves extensive use of solvents and reagents, which can generate harmful pollutants as by-products.

[0008] Producing cells and cellular materials from human cells for human applications is desirable because human cells contain almost everything that human cells need. Although human cell lines could theoretically provide ideal human-origin materials, commercially available lines often present significant challenges. Many human cell lines in the market are derived from cancer patients and / or contain viral transgenes that enable indefinite cell division. These human cell lines may be cancerous in nature and contain viral elements, raising significant safety concerns regarding oncogenicity and pathogenicity.

[0009] Most current products on the market adopt a "one-size-fits-all" approach, designed for the average user. However, individual genetic differences can significantly impact a user's response to the same product, limiting the effectiveness of these formulations. This underscores the need for personalized products that can cater to the unique biological profiles of individual consumers.

[0010] In summary, there remains an unmet need for bioactive materials that can provide maximal efficacy and safety for human applications while addressing environmental sustainability concerns. New approaches are required to overcome the limitations of traditional animal-derived materials, as well as the challenges posed by existing alternatives.Attorney document no.: 233490-10003SUMMARY

[0011] The embodiments of the present disclosure provide methods for preparing cellular materials from human source and compositions of the same for non-therapeutic applications, including but not limited to, dietary supplements, hair care, skin care, cosmetic products, and wound care.

[0012] Aspects of the present invention may provide methods for producing human-sourced cellular materials and the composition thereof for non-therapeutic applications. In some embodiments, the methods comprise cultivating human cells and applying the cellular materials or the derivatives onto a human subject for non-therapeutic purposes. In certain embodiments, the human cells are from commercial human cell lines. In other embodiments, the human cells are directly isolated from the human subject. In some certain embodiments, cellular materials are applied to the same human subject from whom the human cells are obtained. In some embodiments, such non-therapeutic applications include, but are not limited to, skin care, cosmetics, hair care, wound care, and dietary supplements.

[0013] Some embodiments of the present invention may generate cellular materials independent of animal- and plant-derived sources. In some embodiments, this invention offers an environmentally sustainable alternative by minimizing the pollutants generated from the purification of materials sourced from animals and plants. In some embodiments, the present invention aims to produce active ingredients that have a lower likelihood of triggering undesired immune reactions compared to materials derived from non-human species. In further embodiments, the present invention aims to provide active ingredients that are substantially free of pathogens and environmental contaminants typically associated with animal- and plant- derived materials.

[0014] Other embodiments of the present invention may generate cellular materials with improved quality. In some embodiments, this invention aims to deliver active ingredients that exhibit enhanced potency in humans compared to traditional active ingredients produced through recombinant expression, chemical synthesis, or purification from non-human cells or tissues. In other embodiments, the invention seeks to reduce product variability across different batches of the personalized cellular materials by cultivating human cells under a controlled environment.

[0015] Aspects of the present invention may offer the following novel features:

[0016] No reliance on animal, and plant sources;

[0017] No reliance on genetically modified organisms;Attorney document no.: 233490-10003

[0018] Capture of all forms of functional cellular materials naturally produced within cells;

[0019] Multi-functionality and comprehensiveness rather than being limited to single-use, function, action;

[0020] Human-specific origin and product personalization; and

[0021] Optimal safety, efficacy, quality, purity, and outcomes.

[0022] According to one embodiment of the present disclosure, a method for producing and applying cellular materials from human cells for non-therapeutic applications may be provided. The method comprises the steps of obtaining human cells, cultivating the human cells in a controlled environment, harvesting the cultivated human cells, processing the cultivated human cells to produce cellular materials and applying the cellular materials to a human subject for non-therapeutic applications.

[0023] According to another embodiment of the present disclosure, provided herein is a composition comprising cellular materials derived from cultivated human cells for non- therapeutic applications on a human subject.Attorney document no.: 233490-10003BRIEF DESCRIPTION OF DRAWINGS

[0024] The disclosure may be better understood by reference to the detailed description when considered in connection with the accompanying drawings. The components in the figures are not necessarily to scale, emphasis instead being placed upon illustrating the principles of the disclosure.

[0025] FIG. 1 is a flowchart illustrating the sources, forms and applications of cultivated human cells, according to one embodiment of the present disclosure.

[0026] FIG. 2 is a Western blot analysis of the total cell lysate derived from HaCaT human epidermal keratinocyte line, detecting the presence of a) fibulin 1, b) fibulin 5, c) CTGF, and d) chondroitin sulfate.DETAILED DESCRIPTION

[0027] As used herein and in the claims, the terms “comprising” (or any related form such as “comprise” and “comprises”), “including” (or any related forms such as “include” or “includes”), “containing” (or any related forms such as “contain” or “contains”), means including the following elements but not excluding others. It shall be understood that for every embodiment in which the term “comprising” (or any related form such as “comprise” and “comprises”), “including” (or any related forms such as “include” or “includes”), or “containing” (or any related forms such as “contain” or “contains”) is used, this disclosure / application also includes alternate embodiments where the term “comprising”, “including,” or “containing,” is replaced with “consisting essentially of’ or “consisting of’. These alternate embodiments that use “consisting of’ or “consisting essentially of’ are understood to be narrower embodiments of the “comprising”, “including,” or “containing,” embodiments.

[0028] For the sake of clarity, “comprising”, including, and “containing”, and any related forms are open-ended terms which allow for additional elements or features beyond the named essential elements, whereas “consisting of’ is a closed-end term that is limited to the elements recited in the claim and excludes any element, step, or ingredient not specified in the claim.

[0029] For the sake of clarity, “characterized by” or “characterized in” (together with their related forms as described above), does not limit or change the nature of whether the list of terms following it is open or closed. For example, in a claim directed towards “a composition comprising A, B, C, and characterized in D, E, and F”, the elements D, E, and F are still open-Attorney document no.: 233490-10003 ended terms and the claim is meant to include other elements due to the use of the word “comprising” earlier in the claim.

[0030] As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Where a range is referred to in the specification, the range is understood to include each discrete point within the range. For example, 1-7 means 1, 2, 3, 4, 5, 6, and 7.

[0031] As used herein, the term “non-therapeutic” describes the use or applications that do not involve the treatment or prevention of diseases or medical conditions. Non-therapeutic applications include, but are not limited to, uses in wound care, skin care, hair care, dietary supplements, and cosmetics.

[0032] Cell cultivation, more commonly known as cell culture, refers to the methods and processes used to grow and maintain cells in a controlled environment outside their natural setting. This includes the use of specific culture media, growth conditions, and equipment to support cell proliferation, differentiation, and function. The technology encompasses various techniques such as aseptic handling, media preparation, and the use of bioreactors or other specialized devices to optimize cell growth and productivity.

[0033] Aspects of the invention may provide methods of producing and the compositions of non-therapeutic, human-sourced cellular materials offer numerous benefits over existing solutions.

[0034] First, embodiments of the invention facilitate the collection of all functional proteins from intact human cells or from the lysis or hydrolysis of cultivated cells. It employs different forms of cellular materials, including whole cells, total lysates, hydrolysates, and targeted proteins, derived from commercial or personalized human cell lines. Unlike traditional recombinant expression and chemical synthesis methods, this invention ensures that no active components are lost.

[0035] Second, active ingredients produced by embodiments of the current invention are less likely to elicit undesired immune reactions compared to those derived from non-human species. Further, the human cells are cultivated in a controlled environment that is free of pathogens, heavy metals, antibiotics and micro-plastic. The compositions derived therefrom offer high quality and consistency, and can effectively meet human biological needs for growth factors, cytokines, and extracellular matrix proteins etc.

[0036] Third, embodiments of the invention adopt an environmentally sustainable approach by reducing reliance on animal- and plant-derived materials, thereby minimizing pollutants associated with traditional purification methods. The process avoids the use of harmfulAttorney document no.: 233490-10003 chemicals and reduces purification steps, while preserving the functionality of active ingredients and ensuring a cleaner production process.

[0037] Fourth, the active ingredients generated herein exhibit higher potency compared to traditional methods that rely on recombinant expression, chemical synthesis or non-human sources. Human cell-derived actives demonstrate superior bioactivity, allowing for lower quantities to achieve the same or improved effects. Embodiments of the invention may include methods that may capture a broad spectrum of active compounds, minimizing the loss of actives that often occurs during purification steps for conventional products.

[0038] Fifth, functional ingredients generated from cultivated human cells ensure compatibility in post-translational modifications and protein folding, which are crucial for eliciting desired functional effects. This comprehensive collection of actives from the entire cell maximizes efficacy for human users.

[0039] Sixth, embodiments of the present invention are devoid of the use of cancer cell lines, as is common in existing methods. By employing healthy human cells, the current invention prevents the risk of transfer of oncogenic cells and infectious materials from commercial cell lines, addressing concerns related to safety, potential oncogenicity, pathogenicity and compatibility.

[0040] Lastly, embodiments of the present invention allow for the cultivation of individualspecific cells, enabling the application of tailored products that account for genetic variations and receptor differences among users. This personalized approach significantly reduces the risk of adverse reactions and enhances efficacy, in contrast to the conventional one-size-fits-all products.

[0041] Although the description referred to particular embodiments, the disclosure should not be construed as limited to the embodiments set forth herein.

[0042] According to one embodiment of the present disclosure, a method for producing and applying cellular materials from human cells for non-therapeutic applications is provided. The method comprises the steps of obtaining human cells, cultivating the human cells in a controlled environment, harvesting the cultivated human cells, processing the cultivated human cells to produce cellular materials and applying the cellular materials to a human subject for non-therapeutic applications.

[0043] In some embodiments, the human cells are obtained from human cell lines. In certain embodiments, the human cell lines suitable for this invention can be derived from various human organs, including but not limited to, umbilical cord, placenta, skin, heart, lung, liver, kidney and blood. In certain embodiments, the human cell lines are selected from a groupAttorney document no.: 233490-10003 consisting of fibroblast cell lines, including but not limited to, Hs 726. Pl, Hs 795. Pl, BJ, HFF- 1, and CCD-1141 Sk; keratinocyte cell lines, including but not limited to, HaCaT; epithelial cell lines, melanocyte cell lines; endothelial cell lines, including but not limited to, Human Umbilical Vein Endothelial Cells (HUVEC); and embryonic stem cell lines, including but not limited to WA01, WA09-PCBC, and UC06.

[0044] In another embodiment, the human cells are isolated from a human subject. In further embodiments, the process further comprises the step of isolating the human cells from the human subject.

[0045] In some embodiments, the cellular materials are selected from the group consisting of whole cells, total cell lysate, hydrolysate, cell fractions, or purified proteins.

[0046] In some embodiments, the non-therapeutic applications are selected from a group consisting of dietary supplements, hair care products, skin care products, cosmetic formulations, and wound care treatments.

[0047] In certain embodiments, the controlled environment comprises Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% (v / v) fetal bovine serum inside a humidified incubator maintained at 34°C with 5% CO2.

[0048] In some embodiments, the cellular materials comprise at least one of the following: fibulin 1, fibulin 5, connective tissue growth factor (CTGF), and chondroitin sulfate.

[0049] According to another embodiment of the present disclosure, provided herein is a composition comprising cellular materials derived from cultivated human cells. In some embodiments, the composition is formulated for non-therapeutic applications on a human subj ect.

[0050] In certain embodiments, the cellular materials are selected from the group consisting of whole cells, total cell lysate, hydrolysate, cell fractions, or purified proteins.

[0051] In some embodiments, the human cells are derived from human cell lines. In certain embodiments, the human cell lines suitable for this invention can be derived from various human organs, including but not limited to, umbilical cord, placenta, skin, heart, lung, liver, kidney and blood. In certain embodiments, the human cell lines are selected from a group consisting of fibroblast cell lines, including but not limited to, Hs 726. Pl, Hs 795. Pl, BJ, HFF- 1, and CCD-1141 Sk; keratinocyte cell lines, including but not limited to, HaCaT; epithelial cell lines, melanocyte cell lines; endothelial cell lines, including but not limited to, Human Umbilical Vein Endothelial Cells (HUVEC); and embryonic stem cell lines, including but not limited to WA01, WA09-PCBC, and UC06.Attorney document no.: 233490-10003

[0052] In another embodiment, the composition is a personalized formulation, and the human cells are isolated from the human subject.

[0053] In some embodiments, the non-therapeutic applications are selected from a group consisting of dietary supplements, hair care products, skin care products, cosmetic formulations, and wound care treatments.

[0054] In some embodiments, the composition comprises at least one of the following: fibulin 1, fibulin 5, connective tissue growth factor (CTGF), and chondroitin sulfate.

[0055] As an example, FIG. 1 depicts the sources, forms and applications of the cultivated human cells in the present invention.

[0056] According to FIG. 1, the cultivated human cells can be obtained from human cell lines and direct isolation from human individuals. These cultivated human cells can take various forms: whole cells, total lysates, purified protein and hydrolysate. Depending on the form, the cellular materials have various applications. Whole cells can be used in wound healing medical devices (such as artificial skin), food ingredients, and supplements. Total lysates can be used in wound healing medical devices (such as gauze, bandages, saline) and supplements. Purified proteins (such as growth factors) can be used in wound healing medical devices, pharmaceutical products, and topical applications. Hydrolysates (such as Zellulin, and multi-peptides) can also be used in wound healing medical devices and topical applications.EXAMPLES

[0057] Provided herein are examples that describe in more detail certain embodiments of the present disclosure. The examples provided herein are merely for illustrative purposes and are not meant to limit the scope of the invention in any way. All references given below and elsewhere in the present application are hereby included by reference.

[0058] Example 1

[0059] This study illustrates an example method of the current invention to prepare total cell lysate from HaCaT human epidermal keratinocyte line. The composition of the resulting lysate was analyzed.

[0060] Methods

[0061] a. Culture of HaCaT cells

[0062] The HaCaT human epidermal keratinocyte line (Cytion catalog number 300493) was cultured in DMEM supplemented with 10% (v / v) fetal bovine serum inside a humidified incubator at 34°C and 5% CO2. Cells upon confluence were split at a ratio of 1 : 10 for routine maintenance.Attorney document no.: 233490-10003

[0063] b. Preparation of total cell lysate

[0064] The HaCaT cells were harvested at 500 * g for 5 minutes at room temperature. The supernatant was removed. The cell pellet was lysed with RIPA lysis buffer with IX protease inhibitor cocktail for 20 minutes on ice. The lysate was centrifuged at 12,000 * g for 10 minutes at 4°C. The supernatant was collected for use. The protein concentration was quantified by BCA assay according to the manufacturer's instructions.

[0065] c. Western blotting

[0066] The total cell lysate was mixed with 6X Laemmli protein sample buffer supplemented with a reducing agent. The protein sample was denatured at 95°C for 5-10 min. 20 pg total protein and the prestained protein ladder (Thermofisher Scientific) were loaded onto a Novex™ WedgeWell™ 4-20% Tris-Glycine polyacrylamide gel (Thermofisher Scientific). The polyacrylamide gel was run in IX Tris-Glycine SDS Running Buffer (Thermofisher Scientific) at 120V for 75 min. The protein was transferred overnight onto a 0.22 um PVDF membrane (Advansta). The PVDF membrane was blocked in 5% milk in IX TBST for 2 hours. The PVDF membrane was incubated with mouse antibodies against chondroitin sulfate (Abeam), rabbit antibodies against fibulin 1 (Novus), fibulin 5 (Proteintech) and CTGF (Abeam) (1 : 1500 diluted in TBST) for 2 hours at room temperature with constant shaking. The membrane was washed for 5 min for three times. The PVDF membrane was incubated with goat anti-mouse IgG, IgM (H+L) Secondary Antibody, HRP (Thermofisher Scientific) and goat anti-Rabbit IgG (H+L) HRP (Thermofisher Scientific) (1 :2000 diluted in TBST) for 1 hour at room temperature with constant shaking. The membrane was washed for 5 min for three times. The WesternBright ECL components were mixed in a 1 : 1 ratio (Advansta). The PVDF membrane was incubated with the WesternBright ECL mixture for 1 min. The excess WesternBright ECL mixture was removed from the PVDF membrane. The signal was captured using the iBright™ CL1500 Imaging System.Results

[0067] The expression levels of fibulin 1, fibulin 5, CTGF, and chondroitin sulfate were examined in the HaCaT human epidermal keratinocyte cell line using Western blot analysis. The results of such Western blot analysis are shown in FIG. 2. As seen in the figure, distinct bands representing each of a) fibulin 1, b) fibulin 5, c) CTGF, and d) chondroitin sulfate were detected.

[0068] Discussion

[0069] Fibulin 1, fibulin 5, CTGF, and chondroitin sulfate are biomolecules with potential applications in wound healing, skincare, cosmetics, hair care, and dietary supplements. Fibulin-Attorney document no.: 233490-100031 and fibulin-5 are extracellular matrix (ECM) proteins that contribute to tissue structure and functionality. Fibulin-1 is involved in cell adhesion, migration, and the regulation of ECM composition. Fibulin-5, on the other hand, is recognized for its role in elastic fiber formation and its implications for vascular health and skin elasticity. CTGF is a protein that facilitates the actions of various growth factors and is involved in ECM production and fibrosis. It has been studied for its ability to promote ECM production and tissue repair, particularly in the context of wound healing. Chondroitin sulfate is a glycosaminoglycan found in cartilage and connective tissues, where it helps maintain structural integrity and elasticity.

[0070] This study confirms the presence of these biomolecules in the total cell lysate prepared from the HaCaT cell line using the example method.

[0071] The exemplary embodiments of the present invention are thus fully described. Although the description referred to particular embodiments, it will be clear to one skilled in the art that the present invention may be practiced with variation of these specific details. Hence this invention should not be construed as limited to the embodiments set forth herein.

Claims

Attorney document no.: 233490-10003CLAIMSWhat is claimed is:

1. A method for producing and applying cellular materials from human cells for non- therapeutic applications, comprising the steps of : a) obtaining human cells; b) cultivating the human cells in a controlled environment; c) harvesting the cultivated human cells; d) processing the cultivated human cells to produce cellular materials; e) applying the cellular materials to a human subject for non-therapeutic applications.

2. The method of claim 1, wherein the human cells are obtained from human cell line.

3. The method of claim 2, wherein the human cell line is selected from a group consisting of fibroblast cell line, keratinocyte cell line, epithelial cell line, melanocyte cell line, endothelial cell line, and embryonic stem cell line.

4. The method of claim 3, wherein the keratinocyte cell line is HaCaT.

5. The method of claim 3, wherein the fibroblast cell line is selected from the group consisting of Hs 726.P1, Hs 795. Pl, BJ, HFF-1, or CCD-1141Sk; the endothelial cell line is Human Umbilical Vein Endothelial Cells (HUVEC); and the embryonic stem cell line is selected from the group consisting of WA01, WA09-PCBC, or UC06.

6. The method of claim 1, wherein the human cells are isolated from said human subject.

7. The method of claim 6, further comprising the step of isolating the human cells from said human subject prior to step a).

8. The method of claim 1, wherein the cellular materials are selected from the group consisting of whole cells, total cell lysate, hydrolysate, cell fractions, or purified proteins.

9. The method of claim 1, wherein the non-therapeutic applications are selected from a group consisting of dietary supplements, hair care products, skin care products, cosmetic formulation and wound care treatments.

10. The method of step 1, wherein the controlled environment comprises Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% (v / v) fetal bovine serum inside a humidified incubator maintained at 34°C with 5% CO2.

11. The method of claim 1, wherein the cellular materials comprise at least one of the following: fibulin 1, fibulin 5, connective tissue growth factor (CTGF), and chondroitin sulfate.Attorney document no.: 233490-1000312. A composition comprising cellular materials derived from cultivated human cells, wherein the composition is formulated for non-therapeutic applications on a human subject.

13. The composition of claim 12, wherein the cellular materials are selected from the group consisting of whole cells, total cell lysate, hydrolysate, cell fractions, or purified proteins.

14. The composition of claim 12, wherein the human cells are derived from human cell line.

15. The composition of claim 14, wherein the human cell line is selected from a group consisting of fibroblast cell line, keratinocyte cell line, epithelial cell line, and melanocyte cell line.

16. The composition of claim 15, wherein the keratinocyte cell line is HaCaT.

17. The composition of claim 15, wherein the fibroblast cell line is selected from the group consisting of Hs 726.P1, Hs 795. Pl, BJ, HFF-1, or CCD-1141Sk; the endothelial cell line is Human Umbilical Vein Endothelial Cells (HUVEC), the embryonic stem cell line is selected from the group consisting of WA01, WA09-PCBC, or UC06.

18. The composition of claim 12, wherein the composition is a personalized formulation and the human cells are isolated from said human subject.

19. The composition of claim 12, wherein the non-therapeutic applications are selected from a group consisting of dietary supplements, hair care products, skin care products, cosmetic formulation and wound care treatments.

20. The composition of claim 12, wherein the composition comprises at least one of the following: fibulin 1, fibulin 5, connective tissue growth factor (CTGF), and chondroitin sulfate.

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