TW202342735A - Methods of producing human mesenchymal stem cells from foreskins and uses thereof - Google Patents

Abstract

Disclosed herein are methods for providing human mesenchymal stem cells (hMSCs). The methods include steps of, incubating a foreskin tissue in a first culture medium which does not contain serum; cutting the foreskin tissue and treating it with digestive enzymes; pouring the foreskin tissue into a cell strainer; next, placing the cell strainer on a first culture plate and incubating the cell strainer together with the first culture plate at 37° C for at least 12 hours, wherein the first culture plate has a second culture medium with serum; and collecting the human mesenchymal stem cells on the first culture plate.

Description

Methods for obtaining human mesenchymal stem cells from foreskin tissue and their uses

The present disclosure relates to a method of isolating and expanding mesenchymal stem cells (MSCs) and cell populations derived therefrom.

Mesenchymal stem cells have good differentiation ability and can be further induced to differentiate into bone cells, chondrocytes and adipocytes. They are one of the main cells used in regenerative medicine materials. Furthermore, mesenchymal stem cells have anti-inflammatory and immune-regulating effects and are widely used in clinical treatment of diseases.

Mesenchymal stem cells can currently be isolated from various tissues, including but not limited to neonatal tissues (for example: Wharton's jelly of human umbilical cord), bone marrow, peripheral blood, placenta, umbilical cord blood adipose tissue, teeth, etc., but The effect of isolating and proliferating mesenchymal stem cells during the preparation process is limited. It usually takes 10-30 days to culture a sufficient amount of mesenchymal stem cells for subsequent differentiation and related applications.

Therefore, there is an urgent need to develop a novel method for preparing mesenchymal stem cells in related fields to prepare mesenchymal stem cells sufficient for subsequent therapeutic use.

This summary is intended to provide a simplified summary of the disclosure to provide the reader with a basic understanding of the disclosure. This summary is not an extensive overview of the disclosure and it is not intended to identify key/critical elements of the embodiments of the invention or to delineate the scope of the invention.

The method proposed by the present invention can efficiently isolate and culture human mesenchymal stem cells from foreskin tissue, which greatly improves the shortcomings of the previous technology of time-consuming and limited production capacity of cultivating human mesenchymal stem cells.

Accordingly, a first aspect of the disclosure relates to a method of providing human mesenchymal stem cells. The method consists of the following steps: (a) Place the foreskin tissue in a first culture medium, wherein the first culture medium is a keratinocyte culture medium and does not contain serum; (b) After mincing the foreskin tissue cultured in step (a), treat it with a digestive enzyme at 36-37°C for at least 2 hours; (c) Provide a first culture plate containing a second culture medium, wherein the second culture medium is a keratinocyte culture medium with added serum; (d) Place the minced foreskin tissue processed in step (b) into a filter cup, then place the filter cup in the first culture plate, and place the filter cup together with the first culture plate at 37 Incubate at °C for at least 12 hours; and (e) Collect the human mesenchymal stem cells on the first culture plate; Wherein, the foreskin tissue is resected foreskin tissue.

According to an embodiment of the present invention, the method further includes the following steps: (f) Place the filter cup on a second culture plate, wherein the second culture plate contains a second culture medium, culture the filter cup together with the second culture plate at 37°C for at least 2 hours, and (g) Collect the human mesenchymal stem cells on the second culture plate.

According to a specific implementation of the present invention, the method further includes the steps: (a1) Before performing step (a), clean the foreskin tissue with physiological saline.

According to a specific embodiment, the digestive enzymes in the method are collagenase and protease.

In any of the above embodiments, the second medium used may further include at least 3% (volume %) vitamin C, at least 10% (volume %) serum, at least 1% (volume %) antibiotics and/or Or at least 3% (volume %) N-Acetylcystein.

According to a preferred embodiment of the present invention, the human mesenchymal stem cells obtained by any of the above embodiments show negative reactions to CD34 and CD45 cell surface markers, and show positive reactions to CD73, CD90 and CD 105 cell surface markers.

In addition, the present disclosure also relates to the use of human mesenchymal stem cells or their undifferentiated progeny for the preparation of medicaments that can be used to treat diseases or conditions in individuals in need of treatment.

According to embodiments of the present disclosure, the human mesenchymal stem cells are substantially pure human mesenchymal stem cells.

According to embodiments of the present disclosure, substantially pure human mesenchymal stem cells can be induced to differentiate into chondrocytes, skeletal cells, and adipocytes.

According to embodiments of the present disclosure, diseases or conditions treatable by autologous transplantation of human mesenchymal stem cells are selected from the group consisting of bone or cartilage diseases, neurodegenerative diseases, heart diseases, liver diseases, cancer, autoimmune diseases, Graft versus host disease (GvHD) or wound healing and tissue regeneration disease.

In addition, another aspect of the present invention relates to a method of using human mesenchymal stem cells obtained in any of the above embodiments to treat diseases or conditions in individuals in urgent need of treatment. The treatment methods include administering to an individual an effective amount of human mesenchymal stem cells prepared according to the methods of the present disclosure.

According to embodiments of the present disclosure, an effective amount of human mesenchymal stem cells or their differentiation can be continuously administered to an individual for a period of 3 days or less, a period of 2 days or less, or a period of 1 day or less. offspring.

After referring to the following embodiments, those with ordinary knowledge in the technical field to which the present invention belongs can easily understand the basic spirit and other objectives of the present invention, as well as the technical means and implementation modes adopted by the present invention.

In order to make the description of the present disclosure more detailed and complete, the following provides an illustrative description of the implementation aspects and specific embodiments of the present invention; however, this is not the only form of implementing or using the specific embodiments of the present invention. The embodiments cover features of multiple specific embodiments as well as method steps and their sequences for constructing and operating these specific embodiments. However, other specific embodiments may also be used to achieve the same or equivalent functions and step sequences.

1. Definition

For convenience of explanation, specific terms described in this specification, the examples, and the appended claims are collectively described here. Unless otherwise defined in this specification, the scientific and technical terms used herein have the same meanings as commonly understood and customary by a person of ordinary skill in the art to which this invention belongs.

Unless the context clearly indicates otherwise, when used herein, the singular forms "a", "an" and "the" include the plural form.

The term "stem cell" used in this article is used in a broad sense, and also includes traditional stem cells, progenitor cells, pre-progenitor cells, etc. The term "stem cell" refers to undifferentiated cells that can proliferate and produce more precursor cells. These precursor cells have the ability to produce a large number of mother cells and then produce differentiated or differentiated daughter cells. The daughter cells themselves can be induced to proliferate and produce progeny that subsequently differentiate into one or more mature cell types. The term "stem cell" can also refer to the ability or potential to differentiate into a more specialized or differentiated phenotype under certain circumstances, and under certain circumstances, retain the ability to proliferate in an undifferentiated state. cells.

The term "mesenchymal stem cells (MSCs)" refers to a type of pluripotent stem cells that can differentiate into more than one specific type of connective tissue. The specific type of connective tissue can be adipose tissue, bone tissue, stromal tissue, or cartilage tissue. , elastic tissue and fibrous tissue. For identification purposes, human mesenchymal stem cells can be identified based on the expression of phenotypic markers CD34-, CD45-, CD73+, CD90+, and CD105+, or based on differentiation into specific life-supporting tissues (for example, but not limited to, chondrocytes). , bone cells and adipocytes) to identify.

In the context of cell ontogeny, the adjective "differentiated" is a relative concept. A "differentiated cell" refers to a cell whose developmental path is further forward or downstream than that of another cell to which it is being compared. Thus, stem cells can differentiate into lineage-restricted precursor cells, which in turn can differentiate into other precursor cell types further downstream. This is followed by differentiation into cells in the final stages of differentiation that play characteristic roles in specific tissue types and may or may not retain the ability to proliferate further.

The term "treatment" as used herein refers to obtaining a desired pharmacological and/or physiological effect, such as tissue regeneration. In terms of completely or partially preventing a disease or its symptoms, the aforementioned effects may be preventive; and/or in terms of completely or partially treating a disease and/or causing adverse effects of the disease, the aforementioned effects may be therapeutic. of. "Treatment" as used herein includes preventive (e.g. prophylactic), curative or palliative treatment of disease in a mammal, especially a human; and includes: (1) Preventative (e.g., prophylactic), curative, or palliative treatment of a disease or condition that occurs in an individual who may be susceptible to the disease but has not yet been diagnosed with the disease; (2) suppress a disease (e.g., by terminating the progression of the disease); or (3) alleviating a disease (e.g., reducing symptoms associated with the disease).

The terms "administered, administering" or "transplanting" are used interchangeably herein to refer to any appropriate route of delivery, including but not limited to: intravenous, intramuscular, intraperitoneal , intraarterially, intracranially, or subcutaneously administering the human mesenchymal stem cells of the present invention to the desired site of the individual, where at least a part of the human mesenchymal stem cells are still alive. The period of survival of the cells after administration to an individual can be as short as a few hours, such as 24 hours, to a few days, or as long as a few months.

As used in this disclosure, "an effective amount" refers to the dose of human mesenchymal stem cells that is effective in achieving the desired results for the disease to be treated within a specific required period of time. For example, in the treatment of wounds, agents that effectively promote the regeneration of skin and related soft tissues (i.e., the human mesenchymal stem cells of the present disclosure) are used. An effective amount of a pharmaceutical agent does not necessarily cure a disease or condition but may provide treatment for a disease or condition, such as delaying the onset of the disease or condition, limiting or hindering the onset of the disease or condition, or ameliorating the symptoms of the disease or condition. The specific effective amount or sufficient amount will vary depending on various factors, such as the specific condition being treated, the patient's physiological condition (such as the patient's weight, age, or sex), the species of mammal or animal being treated, the duration of treatment, and concurrent The nature of the course of treatment (if any), and the specific preparations used, etc. The effective amount may be divided into one, two or more doses in a suitable form, whereby administration is administered once, twice or more over a specified period of time.

As used herein, the terms "subject," or "patient" are used interchangeably and mean a mammal (including humans) that can be treated with a compound of the present disclosure. The term "mammal" refers to all members of the class Mammalia, which includes humans, primates, domesticated animals and domestic animals (such as rabbits, pigs, sheep, cattle) and animals kept in captivity in zoos and used for exercise animals or pets; and rodents (such as mice and rats). Furthermore, unless gender is explicitly stated, the term "individual" or "subject" or "patient" includes both male (male) and female (female). Accordingly, the term "individual, subject" or "patient" includes any mammal that would benefit from the treatment methods of the present disclosure. Examples of "individuals, subjects" or "patients" include, but are not limited to, humans, rats, mice, guinea pigs, monkeys, pigs, goats, cattle, horses, dogs, cats, birds, and poultry. In a preferred embodiment, the individual is a human being.

The term "medium" refers to a matrix containing nutrients that can maintain cell viability and support cell proliferation, and is used to maintain a tissue or cell population, or to culture a cell population (eg, a culture matrix). Cell culture media contains any appropriate combination of salts, buffers, amino acids, vitamins, glucose or other carbohydrates, antibiotics, serum or serum substitutes, and other ingredients (such as growth factors). Cell culture media for use with specific cell types is known to those of ordinary skill in the art to which this invention pertains. In an optional embodiment, the culture medium is at least 3% (volume %) vitamin C, at least 10% (volume %) serum, at least 1% (volume %) antibiotics and/or at least 3% (volume %) %) of N-Acetylcystein. In a preferred embodiment, the culture medium is keratinocyte culture medium.

The terms "mobility", "activity" and "mobilization" used herein refer to the process by which cells leave their original niche (such as bone marrow) and enter the bloodstream. The term "mobilization agent" refers to an agent that can cause the aggregates or populations of stem cells (such as MSCs) to lose their adhesion in the peripheral blood and bone marrow stem cell compartments.

2. Isolation and culture of human mesenchymal stem cells derived from foreskin tissue

Mesenchymal stem cells (MSCs) are non-hematopoietic stem cells that can generally be obtained from bone marrow. The methods of isolating and cultivating mesenchymal stem cells in the previous technology mainly come from bone marrow, fat, teeth, umbilical cord, placenta, etc. The methods of the previous technology are time-consuming and usually take at least 10-30 days, and cannot be efficiently produced in a short time. A sufficient amount of cells for therapeutic use. The method for preparing human mesenchymal stem cells proposed by the present invention is fast and low-cost. A certain amount of human mesenchymal stem cells can be cultured within 48 hours, and the cultured stem cells have good efficacy and can be used for autologous treatment in regenerative therapy. Such as wound healing and bone repair and other orthopedic indications. Furthermore, the method for preparing human mesenchymal stem cells proposed by the present invention has good subculture ability, and the cells can be subcultured to at least 30 generations.

Accordingly, a first aspect of the present disclosure relates to a method of providing undifferentiated human mesenchymal stem cells, the method comprising the following steps: (a) Culturing the foreskin tissue in a first medium, wherein the first medium is keratinocyte medium (K-medium) and does not contain serum; (b) After mincing the foreskin tissue cultured in step (a), treat it with a digestive enzyme at 36-37°C for at least 2 hours; (c) Provide a first culture plate containing a second culture medium, wherein the second culture medium is a keratinocyte culture medium with added serum; (d) Place the minced foreskin tissue processed in step (b) into a filter cup, then place the filter cup in the first culture plate, and place the filter cup together with the first culture plate at 37 Incubate at °C for at least 12 hours; and (e) Collect the human mesenchymal stem cells on the first culture plate.

Furthermore, in further embodiments, the method further includes the following steps: (f) Place the filter cup on a second culture plate, wherein the second culture plate contains the second culture medium, and culture the filter cup together with the second culture plate at 37°C for at least 2 hours, and (g) Collect the human mesenchymal stem cells on the second culture plate.

The foreskin tissue used in the present invention is the foreskin tissue removed by individual foreskin surgery. Through the above method, human mesenchymal stem cells can be quickly isolated and cultured in at least 14 hours, for example, at least 14, 15, 16, 17, 18, 19 , 20 hours. In addition, the foreskin tissue is preferably processed in a previous treatment step. Specifically, before performing step (a), the obtained foreskin tissue is washed with physiological saline.

In addition, the first culture medium and the second culture medium suitable for the present invention are keratinocyte culture media, which can be commercial culture media available on the market. In one embodiment, the keratinocyte culture medium may be Keratinocyte-SFM (1X) (Gibco, provided by life technologies).

During the culture process, supplements required for cell metabolism, such as amino acids, vitamins, minerals, and useful proteins (such as transferrin), can be added to the culture medium. The culture medium may also contain antibiotics to prevent yeast, bacterial and fungal infections. Antibiotics can be penicillin, streptomycin, gentamicin, etc. According to an embodiment of the present invention, the first culture medium does not contain serum and additional additives, while the second culture medium may additionally add at least 3% (volume %) vitamin C, 10% (volume %) serum and/or At least 1% (volume %) antibiotic. Furthermore, in a non-limiting embodiment, the second culture medium additionally adds at least 3% (volume %) acetylcysteine (N-Acetylcystein). In optional embodiments, the serum may be from animal or human serum. In a specific embodiment, the serum is from human serum or fetal calf serum. In a non-limiting embodiment, the serum and foreskin tissue may be from the same individual, for example, a circumcised individual. In another embodiment, the serum is from a healthy individual whose serotype is the same as the serotype of the corresponding individual who provided the foreskin tissue.

According to a specific embodiment of the present disclosure, the human mesenchymal stem cells prepared in the present invention exhibit negative staining for hematopoietic stem cell markers CD34 and CD45, and are known to mediate graft-versus-host disease (GvHD). ) also showed negative staining for human leukocyte antigen-antigen D related (HLA-DR) cell surface markers; positive staining for cell surface markers CD44, CD73, CD90, CD 105 and CD 166. Methods for determining the expression of cell surface markers are common knowledge in the art. Examples of such methods include immunological methods (such as FACS) and biochemical methods (such as cell surface labeling by radioactivity, fluorescence, or avidin-biotin). Another possible way is to use magnetic-activated cell sorting (MACS) or immunopanning methods to identify cells.

The cell surface marker expression pattern of human mesenchymal stem cells prepared by the method of the present disclosure proves that these human mesenchymal stem cells are indeed undifferentiated mature human mesenchymal stem cells, which can be used for regenerative therapies requiring autologous stem cell transplantation.

According to embodiments of the present disclosure, human mesenchymal stem cells can be expanded to at least two times, at least four times, at least six times, at least eight times, at least ten times, at least twelve times, at least fifteen times, at least twenty times , at least thirty times, or at least forty times.

According to an embodiment of the present disclosure, the volume ratio of the second medium in the filter cup and the second culture plate is at least 1:10 ⁶ , for example, at least 1:10 ⁶ , 1:10 ⁷ , 1:10 ⁸ , 1 :10 ⁹ , 1:10 ¹⁰ .

Furthermore, the secondary human mesenchymal stem cells prepared through steps (f) and (g) also maintained an undifferentiated state and showed negative staining for hematopoietic stem cell markers CD34 and CD45 and cell surface marker HLA-DR cell surface markers; in addition, At the same time, the cell surface markers CD44, CD73, CD90, CD 105 and CD 166 were positively stained.

The human mesenchymal stem cell culture prepared using the method of the present invention can be used in a fresh state, or can be stored for subsequent use, such as freezing with cryopreservatives (including but not limited to glycerol, dimethylsulfoxide (DMSO), etc.) save.

3. Composition containing human mesenchymal stem cells

The human mesenchymal stem cells of the present disclosure can be provided alone, together with culture media, or combined with pharmaceutically acceptable carriers and other additives that can promote cell engraftment and/or organ function (e.g., immunosuppression) agents, antibiotics and growth factors, etc.). Therefore, the human mesenchymal stem cells of the present disclosure can be administered to an individual in the form of a pharmaceutical composition. In the pharmaceutical composition, the human mesenchymal stem cells can be mixed with a pharmaceutical carrier or diluent, such as with sterile saline and an aqueous buffer. The solution is mixed.

Suitable routes of administration may include, but are not limited to, oral, rectal, transmucosal (e.g., nasal), enteral, or parenteral delivery (including: intramuscular, subcutaneous, intraventricular, intrathecal, intravenous, intraperitoneal, or ocular). intravenous injection).

Alternatively, the pharmaceutical composition may be administered locally rather than systemically. For example, the pharmaceutical composition can be injected directly into the target location (eg, an organ).

In accordance with the present disclosure, pharmaceutical compositions may be prepared in a conventional manner using one or more pharmaceutically acceptable carriers. The appropriate formulation form will depend on the route of administration or administration.

For injection, the human mesenchymal stem cells of the present disclosure can be formulated as an aqueous solution, preferably as a physiologically acceptable buffer (eg, Ringer's solution) or a physiologically acceptable salt solution.

Pharmaceutical compositions suitable for use in the present disclosure include compositions containing an active ingredient (eg, human mesenchymal stem cells) in an amount effective to achieve the intended purpose. Determination of the effective amount is well within the ability of one of ordinary skill in the art, particularly in light of the description of this disclosure.

The pharmaceutical compositions of the present disclosure may be present in the form of a kit, which may contain one or more unit dosage forms containing human mesenchymal stem cells of the invention. The kit may further include labels or packaging accessories on or associated with the kit. The label or packaging may state that the kit is intended to treat specific diseases and/or conditions. Alternatively or additionally, the kit may further comprise a buffer, such as phosphate buffered saline or Ringer's solution. The kit may further include instructions on how to administer the human mesenchymal stem cells to the predetermined target location.

According to embodiments of the present disclosure, a kit may comprise (or at least comprise): (a) a first container containing human mesenchymal stem cells of the invention, and optionally (b) a second container containing a buffer, and ( c) A legend associated with the set to instruct the user on how to use the set. The legend may be in the form of a booklet, tape, CD, VCD or DVD.

4. Use of human mesenchymal stem cells or their differentiated progeny of the present invention

The undifferentiated human mesenchymal stem cells or their differentiated progeny prepared by the method of the present invention can be used for autologous transplantation to treat and/or prevent numerous diseases and/or conditions for which autologous transplantation can obtain beneficial effects.

Accordingly, other aspects of the present disclosure are directed to methods of treating diseases and/or conditions in an individual, comprising administering to the individual, over a period of days to weeks, an effective amount of a drug prepared according to the method of the present invention. Human mesenchymal stem cells.

Mesenchymal stem cells can differentiate into various cell lineages. Cell differentiation can be achieved by culturing mesenchymal stem cells in a growth environment rich in cells with desired phenotypes (such as bone cells, adipocytes, etc.). The culture medium may contain agents that enhance differentiation into specific cell lines. According to one embodiment of the present disclosure, human mesenchymal stem cells are differentiated into chondrocytes, wherein the cells contain dexamethasone, ascorbic acid, insulin, transferrin and selenous acid. acid) medium until confluence (please refer to Williams et al. (2003) Tissue Engineering. 9(4), 679). Differentiated chondrocytes can be identified by Alcian blue and toluidine blue. According to yet another embodiment of the present disclosure, human mesenchymal stem cells are differentiated into adipocytes. To induce adipogenic differentiation, human mesenchymal stem cells are treated with adipogenic medium containing hydrocortisone and indomethacin; alternatively or in addition, any commercially available mesenchymal stem cells containing Lipogenesis promoting supplements (such as those available from StemCell Technologies Inc (Vancouver, Canada)). The results of adipogenic differentiation can be confirmed by oil red staining. Human mesenchymal stem cells can also be induced to form skeletal cells using a commercially available osteogenic differentiation kit. The differentiation results of differentiated skeletal cells can be confirmed by Alizarin Red staining.

According to embodiments of the present disclosure, human mesenchymal stem cells or their differentiated progeny may be used to treat bone or cartilage diseases, neurodegenerative diseases, heart diseases, liver diseases, cancer, autoimmune diseases, graft versus host disease ( GvHD) and the group of diseases and/or conditions that comprise wound healing and tissue regeneration.

Human mesenchymal stem cells or their differentiated progeny prepared by the method of the present invention can be suitable for the treatment of bone defects, which include, but are not limited to: osteogenesis imperfecta (osteogenesis imperfecta), fracture (fracture), congenital bone defects (congenital bone defects) )wait.

Human mesenchymal stem cells or their differentiated progeny obtained through the aforementioned method of the present invention can also be implanted into a body to provide bony and connective support for bones and other artificial devices (such as teeth), such as joint implants and /or dental implants.

Since mesenchymal stem cells can differentiate into cartilage, human mesenchymal stem cells or their differentiated progeny prepared by the method of the present invention can be suitable for the treatment of joint diseases, including but not limited to: osteoarthritis, rheumatoid arthritis Rheumatoid arthritis, inflammatory arthritis, chondromalacia, avascular necrosis, traumatic arthritis, etc.

The human mesenchymal stem cells prepared by the method of the present invention can also be used to treat central nervous system (CNS) diseases. Examples of central nervous system diseases include, but are not limited to: a pain disorder, a movement disorder, a dissociative disorder, a mood disorder, an affective disorder, a neurodegenerative disorder, and A convulsive disorder. More specific examples of such disorders include, but are not limited to: Parkinson's disease, amyotrophic lateral sclerosis (ALS), multiple sclerosis, Henry's disease. Huntington's disease, autoimmune encephalomyelitis, diabetic neuropathy, glaucomatous neuropathy, macular degeneration, action tremor action tremors and tardive dyskinesia, panic, anxiety and depression, alcoholism, insomnia, manic behavior, Alzheimer's disease and epilepsy ).

Mesenchymal stem cells are known to interact with hematopoietic stem cells and immune cells and exhibit cell therapy potential to enhance autologous hematopoietic engraftment and avoid graft-versus-host disease (GvHD). Accordingly, human mesenchymal stem cells prepared by the method of the present invention can also be used to treat GvHD.

The human mesenchymal stem cells or their differentiated progeny prepared by the method of the present invention can also be used to promote tissue regeneration. In this way, the transplantation of human mesenchymal stem cells can be used to treat autoimmune diseases, inflammatory diseases, acute and chronic ischemic diseases, tissue engineering; it can also regenerate new tissues and naturally heal diseased or damaged organs.

It is known that when mesenchymal stem cells are introduced into infarcted hearts, these cells prevent harmful (ventricular) remodeling and enhance recovery. Injecting mesenchymal stem cells directly into the infarcted heart or administering the cells intravenously can return the cells to the site of injury. Accordingly, the human mesenchymal stem cells or their differentiated progeny of the present invention can also be used to treat heart diseases, especially the formation of cardiac infarction.

Examples of cancers that can be treated by human mesenchymal stem cells or their differentiated progeny of the present invention include, but are not limited to, breast cancer, brain tumor, melanoma, lung cancer, lymphoma ( lymphoma), neuroepithelioma, kidney cancer, prostate cancer, stomach cancer, colon cancer, rectal cancer, pancreatic cancer cancer) and uterus cancer. In some embodiments, the cancer is metastatic cancer.

The human mesenchymal stem cells of the present invention, or their differentiated progeny, can be administered to the treated individual using a variety of transplantation methods, with their properties depending on the site of implantation. Cells can be transplanted into damaged or healthy areas of tissue. When human mesenchymal stem cells are administered to healthy areas, these cells will move to damaged areas.

The human mesenchymal stem cells of the present invention or their differentiated progeny can be transplanted by direct injection into an organ, direct injection into the blood stream, intraperitoneal injection, or direct injection into lymphoid organs. Appropriate transplantation methods can be determined by monitoring the return and engraftment of the implanted cells to the intended organ, the expression of the intended organ-specific markers, and the intended organ function of the individual.

An effective amount of human mesenchymal stem cells or their differentiated progeny of the present invention is administered on the premise that the desired therapeutic effect can be achieved in an individual. The effective dose of human mesenchymal stem cells used in the present invention can be preliminarily estimated from in vivo and/or in vitro cell culture assays. For example, doses that achieve desired concentrations can be formulated in experimental animals and then used to more accurately determine doses for humans. Dosage may depend on the administration or route of administration. The exact dosage and route of administration/administration can be determined by the attending physician based on the patient's condition and medical history.

Depending on the condition to be treated, the effective amount of human mesenchymal stem cells of the present invention can be a single dose or multiple doses, and the treatment process can last from days to weeks, or until effective cure or symptoms of the disease are reduced.

Additional objects, advantages and features of the present invention will be apparent to those of ordinary skill in the art through examination of the following non-limiting examples.

Example

Materials and methods

Surface antigen analysis

Harvest cells from cell culture dishes using 0.25% trypsin-EDTA. The cells were washed with phosphate buffered saline (PBS) solution containing 1% BSA, and treated with fluorescein isothiocyanide (FITC) or phycoerythrin (PE)- Stain with complexed antibodies for 30 minutes at 4°C. Mesenchymal stem cell markers: anti-CD 44, anti-CD73, anti-CD 90, anti-CD105, anti-CD166, and hematopoietic stem cell markers: anti-CD34, anti-CD45 (negative control group). The cells were then washed with PBS and analyzed using a flow cytometer (product name: FACS calibur flow cytometer, purchased from FACSCanto, BD Biosciences, Becton, Dickinson and Company, San Jose, CA). Cells are passed through at a rate of up to 1,000 cells/second, and a 488 nm argon laser beam is used as the excitation light source. Cells stained with FITC and PE-complexed isotype control antibodies can be used to calculate background fluorescence values.

Cell differentiation induced

Human mesenchymal stem cells are induced to form bone cells using an osteogenic differentiation kit (trade name: STEM Osteogenesis Differentiation Kit, Gibco), and an adipogenic differentiation kit (trade name: STEMPRO adipogenic differentiation kit, Gibco) and chondrogenic induction medium ( Chondrogenic induction medium, Gibco) induces the formation of adipocytes and chondrocytes. Then, maintain the survival state of these cells in a humidified incubator with 95% air and 5% _CO2 at a temperature of 37°C. The culture medium was changed every 2 to 5 days over a period of 14 days. Alcian blue staining and toluidine blue staining were used to evaluate differentiated chondrocytes; Oil Red-O staining was used to evaluate differentiated adipocytes; and Alzarin Red S was used to evaluate differentiated skeletal cells according to standard procedures.

Example 1: Preparation and culture of human mesenchymal stem cells (hMSCs)

Obtain the excised foreskin tissue. The foreskin tissue may be the foreskin cut off by a circumcision method. Place the foreskin tissue (about 1.5-2 cm wide) into a sterile glass beaker containing physiological saline (50 ml), shake and rinse. Next, the washed foreskin tissue was transferred to keratinocyte culture medium (5 ml) without serum and any additives (i.e., Keratinocyte-SFM (1X); Gibco, provided by life technologies). In a biosafety cabinet (BSC) (laminar flow) with dust falling within 0.5 μm Class 100, cut the foreskin tissue into pieces and treat it with keratinocyte culture medium containing collagenase and protease, and place it at 36-37° C. Incubate in a constant temperature oven for 2 hours, centrifuge at 13 xg for 9 minutes, and remove the supernatant. Add the processed foreskin tissue fragments to the filter cup (sieve mesh size: 70 micromm; diameter 0.5 cm, height 1 cm), place the filter cup on a sterile culture plate (diameter 9 cm), where the sterile The culture plate contains 8 ml of keratinocyte culture medium (containing 10% serum, 3% Vit C, 3% acetyl cysteine and 1% antibiotics), and then incubated at 37°C, humidity 60-70%, and 5% CO ₂ Cultivate in a cell culture incubator for 12 hours until the cells on the sterile culture plate are confluent (greater than 80% confluence), and then mesenchymal stem cells are produced. Next, place the filter cup (70 μm) on the sterile culture plate containing keratinocyte culture medium (containing 10% blood, 3% Vit C, 3% acetyl cysteine) onto another sterile culture plate. acid and 1% antibiotics), culture it in a cell culture incubator with a temperature of 36-37 degrees, a humidity of 60-70%, and 5% CO ₂ , and culture it for 2-3 hours until the cells on the sterile culture plate are full (confluence). (greater than 80% confluence), the human mesenchymal stem cells can be harvested. Repeat the previous steps, and after the culture is completed, move the filter cup to another clean sterile culture plate to efficiently prepare human mesenchymal stem cells. In this way, the method proposed by the present invention can amplify and culture at least 30 plates, preferably at least 40 plates within 24 hours. The cell morphology results of the human mesenchymal stem cells prepared in this example using a phase contrast microscope at 40 times are shown in Figure 1. The photos taken under the microscope show that the mesenchymal stem cells prepared using the method of the present invention Stem cells are in the form of long spindles and are adherent.

Example 2: Characteristic analysis of human mesenchymal stem cells in Example 1

2.1 Cell surface antigen analysis

Use trypsin/EDTA to separate the adherent cells in Example 1, and subject these cells to surface antigen analysis in a flow cytometer according to the process described in the "Materials and Methods" section. The experimental results are shown in Figure 2.

The human mesenchymal stem cells prepared using Example 1 showed positive reactions to the surface markers CD44, CD73, CD90, CD105, and CD166, but showed negative reactions to CD34, CD45, and HLA-DR (Figure 2); these results confirmed These cells were indeed mesenchymal stem cells.

2.2 Differentiation of human mesenchymal stem cells in Example 1

In this example, in order to confirm that the human mesenchymal stem cells of Example 1 still have the ability to differentiate, these cells were cultured in a culture medium containing a reagent that can induce subsequent differentiation of human mesenchymal stem cells. According to standard procedures, the chondrocytes, bone cells and adipocytes differentiated from Example 1 can be confirmed by Alcian blue staining, toluidine blue staining, alizarin red, and Oil Red-O staining respectively. Cell type, the results are shown in Figure 3A, Figure 3B, Figure 3C and Figure 3D respectively.

It should be understood that the foregoing description of the embodiments is given by way of example only, and various modifications may be made by those skilled in the art. The above specification, examples, and experimental results provide a complete description of the structure and uses of exemplary embodiments of the invention. Although the above embodiments disclose various specific embodiments of the present invention, they are not intended to limit the present invention. Those with ordinary knowledge in the technical field to which the present invention belongs can, without departing from the principles and spirit of the present invention, Various changes and modifications can be made to it, so the protection scope of the present invention shall be defined by the appended patent application scope.

without

In order to make the above and other objects, features, advantages and embodiments of the present invention more apparent and understandable, the accompanying drawings are described as follows: Figure 1 is a photo of the cell morphology of mesenchymal stem cells taken under a microscope (40x) according to one embodiment of the present invention; Figure 2 shows the positive expression results of the human mesenchymal stem cells of Example 1 on the markers CD 44, CD73, CD 90, CD105 and CD166, and the negative expression results of the marker CD34 according to one embodiment of the present disclosure. ; Figure 3A is a photograph according to one embodiment of the disclosure, which is the result of Alcian blue staining confirming the differentiation of human mesenchymal stem cells into chondrocytes in Example 1; Figure 3B is a photograph according to one embodiment of the present disclosure, which is the result of confirming the differentiation of human mesenchymal stem cells into chondrocytes in Example 1 by toluidine blue staining; Figure 3C is a photograph according to one embodiment of the present disclosure, which is the result of alizarin red staining to confirm the differentiation of human mesenchymal stem cells into skeletal cells in Example 1; and The 3D picture is a photograph according to one embodiment of the present disclosure, which is the result of confirming the differentiation of human mesenchymal stem cells into adipocytes in Example 1 by Oil Red O staining.

Claims (12)

A method of obtaining human mesenchymal stem cells (mesenchymal stem cells) from a foreskin tissue, including: (a) Place the foreskin tissue in a first medium, wherein the first medium is keratinocyte medium and does not contain serum; (b) After mincing the foreskin tissue cultured in step (a), treat it with a digestive enzyme at 36-37°C for at least 2 hours; (c) Provide a first culture plate containing a second culture medium, wherein the second culture medium is a keratinocyte culture medium with added serum; (d) Place the minced foreskin tissue processed in step (b) into a filter cup, then place the filter cup in the first culture plate, and place the filter cup together with the first culture plate at 37 Incubate at °C for at least 12 hours; and (e) Collect the human mesenchymal stem cells on the first culture plate; Wherein, the foreskin tissue is resected foreskin tissue. The method described in request 1 further includes: (f) Place the filter cup on a second culture plate, wherein the second culture plate contains the second culture medium, and culture the filter cup together with the second culture plate at 37°C for at least 2 hours, and (g) Collect the human mesenchymal stem cells on the second culture plate. The method described in request 1 further includes: (a1) Before performing step (a), clean the foreskin tissue with physiological saline. The method of claim 1, wherein the digestive enzymes are collagenase and protease. The method of claim 1, wherein the second culture medium further contains at least 3% (volume %) vitamin C. The method of claim 5, wherein the second culture medium further contains at least 10% (volume %) serum. The method of claim 5, wherein the second culture medium further contains at least 1% (volume %) antibiotics. The method of claim 5, wherein the second culture medium further contains at least 3% (volume %) acetylcysteine (N-Acetylcystein). The method of any one of claims 1 to 8, wherein the human mesenchymal stem cells exhibit negative reactions to CD34 and CD45 cell surface markers, and exhibit positive reactions to CD73, CD90 and CD 105 cell surface markers. The use of human mesenchymal stem cells or their undifferentiated progeny in the preparation of a medicament that can be used to treat a disease or condition in an individual in urgent need of treatment, wherein the human mesenchymal stem cells are according to claims 1 to 9 prepared by any method. The use as claimed in claim 10, wherein the human mesenchymal stem cells can differentiate into chondrocytes, bone cells or adipocytes. The use as claimed in claim 10, wherein the disease or condition is a bone or cartilage disease, a neurodegenerative disease, a heart disease, a liver disease, a cancer, an autoimmune disease, a graft versus host disease ( graft versus host disease (GvHD) or a wound healing and tissue regeneration disease.