DESC:DESCRIPTION:
Field of the invention:
[0001] The present disclosure generally relates to the technical field of biomedical technology, and in specific relates to, a method that isolates stem cells from menstrual waste for treating metabolic (such as diabetes), cardiovascular, bone, and cartilage disorders.
Background of the invention:
[0002] In general, physiological menopause occurs around the age of 50, and ovarian function declines gradually. Premature ovarian insufficiency (POI) occurs when women have atypical menstrual cycles, FSH levels rise, and oestrogen volatility falls before the age of 40. Premature ovarian failure (POF) is characterized by amenorrhoea, high FSH levels (>40 IU/L), low oestrogen levels, perimenopausal symptoms, and infertility.

[0003] However, with the current trend towards a younger onset of cancer and the widespread use of radiotherapy and chemotherapy, iatrogenic ovarian function damage caused by chemotherapy drugs is becoming a growing concern, particularly in patients who require fertility treatment. The ovaries are extremely susceptible to chemotherapeutic medicines, particularly alkylating agents, resulting in severe gonadal dysfunction.

[0004] Furthermore, paediatric cancer study indicates that among survivors who received alkylating drugs in combination with abdomen ionizing radiation, the cumulative prevalence of premature menopause was about 20%. As a result, while tumor therapy can improve patients' survival rates, the consequences of ovarian malfunction include early menopause and loss of fertility, which are directly associated with the onset of hot flashes, osteoporosis, and cardiovascular disease.

[0005] Currently, the primary treatment for POF is hormone replacement therapy. However, HRT can only treat perimenopausal symptoms and cannot completely prevent POF. Furthermore, long-term use of exogenous hormones might considerably raise the risk of thrombotic disorders and tumors. Furthermore, infertile patients' reproductive needs can only be provided by treatments such as oocyte cryopreservation, embryo transplantation, and ovarian tissue cryopreservation, and prolonging cancer treatment to retain fertility and obtain oocytes is deemed unethical.

[0006] Fortunately, stem cell-based therapeutics show promise in illnesses that now lack effective treatments. A variety of stem cells, including induced pluripotent stem cells (iPSCs), mesenchymal stem cells (MSCs), and endothelial progenitor cells (EPCs), particularly MSCs, have been used to treat animal models with chemotherapy-induced POI, with promising results observed following stem cell transplantation. Current technology allows for the creation of a human endometrial (menstrual blood) stem cell bank based on female menstruation.

[0007] First, stem cells are harvested and transferred. The transferred cells' data are then collected, allowing the quality of stem cells to be assessed. Later, the stem cells are preserved in a freezer. Compared to other stem cells, stored human endometrium stem cells have the advantages of converting waste into wealth, being high in content, safe and painless to collect, high in the adaptability of people, rich in resources, high in differentiation potential, free of ethical arguments in transplantation, and easy and quick to grow. However, the human endometrial (menstrual blood) stem cell storage bank creation does not effectively treat medical problems.

[0008] Therefore, there is a need for a method that isolates stem cells from menstrual waste for treating various medical disorders. There is also a need for a method that is cost-effective so that the repurposing of endometrial stem cells gets high demand. There is also a need for a method that is an economic source, which utilizes a non-invasive approach that is not been explored in detail. Further, there is also a need for a method that has high potential in making stem cells much more available for stem cell therapy especially to treat bone and cartilage disorders.
Objectives of the invention:
[0009] The primary objective of the invention is to provide a method that isolates stem cells from menstrual waste for treating metabolic (such as diabetes), cardiovascular, bone and cartilage disorders.

[0010] Another objective of the invention is to provide a method that is cost-effective so that the repurposing of endometrial stem cells gets high demand.

[0011] The other objective of the invention is to provide a method that is an economic source, which utilizes a non-invasive approach that is not been explored in detail.

[0012] The other objective of the invention is to provide a method that repurposes endometrial stem cells to cure a wide range of disorders, which could not be possible with conventional treatments.

[0013] Yet another objective of the invention is to provide a method that has high potential in making stem cells much more available for stem cell therapy especially to treat bone and cartilage disorders.

[0014] Further objective of the invention is to provide a method that develops organoids of various types for multiple industrial applications.
Summary of the invention:
[0015] The present disclosure proposes a method for repurposing endometrial stem cells isolated from menstrual waste. The following presents a simplified summary in order to provide a basic understanding of some aspects of the claimed subject matter. This summary is not an extensive overview. It is not intended to identify key/critical elements or to delineate the scope of the claimed subject matter. Its sole purpose is to present some concepts in a simplified form as a prelude to the more detailed description that is presented later.

[0016] In order to overcome the above deficiencies of the prior art, the present disclosure is to solve the technical problem to provide a method that isolates stem cells from menstrual waste for treating various medical disorders.

[0017] According to an aspect, the invention provides a method for repurposing endometrial stem cells isolated from menstrual waste. At one step, menstrual blood waste is collected from a healthy female donor and an equal amount of phosphate-buffered saline (PBS) is added to the menstrual blood waste to form a first mixture. At one step, the first mixture is centrifuged at a speed of at least 5000 rpm for a time period of at least 10 min to obtain a buffy coat and the buffy coat is transferred to a separate tube.

[0018] At one step, the phosphate-buffered saline (PBS) is added to the buffy coat to form a second mixture and centrifuged to form a third mixture. In particular, the second mixture is centrifuged at the speed of at least 1000 rpm for the time period of at least 7 min. At one step, supernatant is discarded from the third mixture and the resulting pellet is washed for at least two times with the PBS. At one step, pellet is suspended in a Dulbecco's Modified Eagle Medium (DMEM) growth medium that contains 10 % of fetal bovine serum (FBS) and the pellet is incubated at a temperature of at least 37 °C in culture flasks. The DMEM growth medium includes anti-mycotic and antibacterial solutions to prevent contamination during stem cell culture.

[0019] At one step, stem cells are isolated from the menstrual waste and the stem cells are cultured in the DMEM growth medium with at least 10 % of fetal bovine serum (FBS) in the presence of glucose. At one step, the stem cells are subjected to glucose starvation and the stem cells are tested for insulin production and other biomarkers to confirm their differentiation into insulin-producing cells. The glucose starvation enhances the metabolic adaptation of the stem cells, thereby enhancing their differentiation into insulin-producing cells. The stem cells are tested for specific biomarkers associated with insulin production to confirm their functional characteristics.

[0020] The stem cells are more accessible and economical than other approaches, which are available in the market that produce insulin. The insulin-producing cells are tested for specific biomarkers associated with insulin production to confirm their functional characteristics. The method requires less investment, thereby resulting in cheaper production of safe insulin.

[0021] Further, objects and advantages of the present invention will be apparent from a study of the following portion of the specification, the claims, and the attached drawings.
Detailed description of drawings:
[0022] The accompanying drawings, which are incorporated in and constitute a part of the specification, illustrate an embodiment of the invention, and, together with the description, explain the principles of the invention.

[0023] FIG. 1 illustrates a flowchart of a method for repurposing endometrial stem cells isolated from menstrual waste, in accordance to an exemplary embodiment of the invention.

[0024] FIG. 2 illustrates a schematic representation of a process for repurposing endometrial stem cells isolated from menstrual waste, in accordance to an exemplary embodiment of the invention.
Detailed invention disclosure:
[0025] Various embodiments of the present invention will be described in reference to the accompanying drawings. Wherever possible, same or similar reference numerals are used in the drawings and the description to refer to the same or like parts or steps.

[0026] The present disclosure has been made with a view towards solving the problem with the prior art described above, and it is an object of the present invention to provide a method that isolates stem cells from menstrual waste for treating bone and cartilage disorders.

[0027] Insulin is often created using recombinant DNA technology, which involves a synthesis of the gene sequence that codes for insulin protein, followed by cloning and expression of that gene. However, various techniques of producing insulin are currently being investigated, taking into account considerations such as the procedure involved vs. the end output.

[0028] In certain investigations, pluripotent stem cells from bone marrow and induced pluripotent stem cells are employed in 2D and 3D cultures to manufacture insulin in response to a glucose challenge. Isolated pancreatic islet cells are also being cultured to produce insulin-secreting organoids. However, the practicality and accessibility of pancreatic stem cells and islet cells continue to be key barriers. The proposed method isolates stem cells from menstrual waste for treating metabolic disorders such as diabetes.

[0029] According to an exemplary embodiment of the invention, FIG. 1 refers to a flowchart 100 of a method for repurposing endometrial stem cells isolated from menstrual waste. At step 102, menstrual blood waste is collected from a healthy female donor and an equal amount of phosphate-buffered saline (PBS) is added to the menstrual blood waste to form a first mixture. At step 104, the first mixture is centrifuged at a speed of at least 5000 rpm for a time period of at least 10 min to obtain a buffy coat and the buffy coat is transferred to a separate tube.

[0030] At step 106, the phosphate-buffered saline (PBS) is added to the buffy coat to form a second mixture and centrifuged to form a third mixture. In particular, the second mixture is centrifuged at the speed of at least 1000 rpm for the time period of at least 7 min. At step 108, supernatant is discarded from the third mixture and the resulting pellet is washed for at least two times with the PBS. At step 110, pellet is suspended in a Dulbecco's Modified Eagle Medium (DMEM) growth medium that contains 10 % of fetal bovine serum and the pellet is incubated at a temperature of at least 37 °C in culture flasks. The DMEM growth medium includes anti-mycotic and antibacterial solutions to prevent contamination during stem cell culture.

[0031] At step 112, stem cells are isolated from the menstrual waste and the stem cells are cultured in the DMEM growth medium with at least 10 % of fetal bovine serum (FBS) in the presence of glucose. At step 114, the stem cells are subjected to glucose starvation and the stem cells are tested for insulin production and other biomarkers to confirm their differentiation into insulin-producing cells. The glucose starvation enhances the metabolic adaptation of the stem cells, thereby enhancing their differentiation into insulin-producing cells.

[0032] The stem cells are tested for specific biomarkers associated with insulin production to confirm their functional characteristics. The stem cells are more accessible and economical than other approaches, which are available in the market that produce insulin. The insulin-producing cells are tested for specific biomarkers associated with insulin production to confirm their functional characteristics. The method requires less investment, thereby resulting in cheaper production of safe insulin.

[0033] In one embodiment herein, the endometrial stem cells can have the capacity to differentiate into almost any cell in the human body, have high potential to cure several disorders in humans. Obtaining viable adult stem cells from sources like bone marrow may be an invasive process and is considered highly expensive. The proposed method is a non-invasive process. The proposed method greatly revolutionizes stem cell therapy by making endometrial stem cells available to treat bone and cartilage disorders. The endometrial stem cells are in great demand because they can treat a wide range of disorders.

[0034] According to another embodiment of the invention, FIG. 2 refers to a schematic representation 200 of a process for repurposing endometrial stem cells isolated from menstrual waste. At step 202, the menstrual blood waste is collected from the healthy female donor. The collection is performed in a sterile manner to minimize contamination. At step 204, the equal volume of phosphate-buffered saline (PBS) is added to the menstrual blood waste to create a first mixture. PBS serves as an isotonic buffer that helps to maintain the physiological pH and osmotic balance of the sample during subsequent processing steps.

[0035] At step 206, the first mixture is subjected to centrifugation at a speed of at least 5000 revolutions per minute (rpm) for a minimum duration of 10 minutes. At step 208, this high-speed centrifugation allows for the separation of cellular components based on their density, leading to the formation of a buffy coat layer. The buffy coat, which contains a rich concentration of leukocytes (white blood cells) and other cellular elements, is carefully transferred to a separate tube for further processing. PBS is added to the buffy coat to create a second mixture. This dilution is essential for washing the cellular components and ensuring optimal separation during centrifugation.

[0036] The second mixture is then centrifuged at a speed of at least 1000 rpm for a duration of at least 7 minutes. This step further separates the cells from any remaining plasma and debris, resulting in a third mixture. At step 210, the supernatant (the liquid above the pellet) from the third mixture is carefully discarded to leave behind the cell pellet. The resulting pellet is washed with PBS at least two times. This washing process is critical for removing any residual plasma proteins, platelets, and other contaminants, thus enhancing the purity of the pellet. After the washes, a clean, purified pellet is obtained, ready for the next steps in cell culture.

[0037] At step 212, the purified pallet is suspended in Dulbecco's Modified Eagle Medium (DMEM) growth medium that contains 10 % of fetal bovine serum (FBS). The DMEM medium is a nutrient-rich solution formulated to support the growth of mammalian cells. To further ensure cell viability and prevent contamination, the medium is supplemented with anti-mycotic and antibacterial solutions. The suspension is incubated at a controlled temperature of at least 37 °C in culture flasks, providing an optimal environment for cell growth. The stem cells are isolated from the menstrual waste contained in the DMEM growth medium. The isolated stem cells are cultured in the DMEM medium enriched with at least 10% fetal bovine serum (FBS), which supplies essential growth factors, hormones, and nutrients necessary for stem cell proliferation.

[0038] Glucose is also present in the medium, serving as a primary energy source for cellular metabolism. At step 214, the stem cells are subjected to glucose starvation, a technique that alters cellular metabolic pathways to promote differentiation. During this phase, the stem cells are closely monitored and tested for insulin production and other specific biomarkers associated with the differentiation of stem cells into insulin-producing cells. The glucose starvation enhances the metabolic adaptation of the stem cells, facilitating their maturation into functional insulin-secreting cells.

[0039] The cells are assessed for biomarkers such as insulin, C-peptide, and specific transcription factors indicative of pancreatic beta cell differentiation, confirming their functional characteristics. This elaborated procedure outlines the meticulous steps involved in isolating and culturing endometrial stem cells from menstrual waste. Each step is designed to ensure high purity and viability of the stem cells, culminating in their differentiation into insulin-producing cells through a carefully controlled environment. The inclusion of detailed parameters, such as centrifugation speeds and incubation conditions, underscores the technical rigor of the process, making it suitable for application in research and therapeutic contexts.

[0040] Numerous advantages of the present disclosure may be apparent from the discussion above. In accordance with the present disclosure, a method for repurposing endometrial stem cells isolated from menstrual waste is disclosed. The proposed method isolates stem cells from menstrual waste for treating metabolic disorders. The proposed method is cost-effective so the repurposing of endometrial stem cells is in high demand. The proposed method is an economic source, which utilizes a non-invasive approach that is not been explored in detail. The proposed method repurposes endometrial stem cells to cure a wide range of disorders, which could not be possible with conventional treatments. The proposed method has high potential in making stem cells much more available for stem cell therapy especially to treat bone and cartilage disorders. The proposed method develops organoids of various types for multiple industrial applications.

[0041] It will readily be apparent that numerous modifications and alterations can be made to the processes described in the foregoing examples without departing from the principles underlying the invention, and all such modifications and alterations are intended to be embraced by this application.
,CLAIMS:CLAIMS:
I / We Claim:
1. A method for repurposing endometrial stem cells isolated from menstrual waste, comprising:
collecting menstrual blood waste from a healthy female donor and adding an equal amount of phosphate-buffered saline (PBS) to the menstrual blood waste to form a first mixture;
centrifuging the first mixture to obtain a buffy coat and transferring the buffy coat to a separate tube;
adding the phosphate-buffered saline (PBS) to the buffy coat to form a second mixture and centrifuging it to form a third mixture;
discarding supernatant from the third mixture and washing the resulting pellet for at least two times with the PBS;
suspending pellet in a Dulbecco's Modified Eagle Medium (DMEM) growth medium that contains at least 10 % of feral bovine serum (FBS) and incubating the pellet in culture flasks;
isolating adherent stem cells from the menstrual waste and culturing the stem cells in the DMEM growth medium the at least 10 % of fetal bovine serum (FBS) in the presence of glucose; and
subjecting the stem cells to glucose starvation and testing the stem cells for insulin production and other biomarkers to confirm their differentiation into insulin-producing cells.
2. The method as claimed in claim 1, wherein the first mixture is centrifuged at a speed of at least 5000 rpm for a time period of at least 10 min.
3. The method as claimed in claim 1, wherein the second mixture is centrifuged at the speed of at least 1000 rpm for the time period of at least 7 min.
4. The method as claimed in claim 1, wherein the DMEM growth medium includes anti-mycotic and antibacterial solutions to prevent contamination during stem cell culture.
5. The method as claimed in claim 1, wherein the pellet is incubated at a temperature of at least 37 °C in culture flasks.
6. The method as claimed in claim 1, wherein the glucose starvation enhances the metabolic adaptation of the stem cells, thereby enhancing their differentiation into insulin-producing cells.
7. The method as claimed in claim 1, wherein the insulin-producing cells are tested for specific biomarkers associated with insulin production to confirm their functional characteristics.
8. The method as claimed in claim 1, wherein the stem cells are tested for specific biomarkers associated with insulin production to confirm their functional characteristics.
9. The method as claimed in claim 1, wherein the method requires less investment, thereby resulting in cheaper production of safe insulin.
10. The method as claimed in claim 1, wherein the stem cells are more accessible and economical than other approaches, which are available in the market that produce insulin.