Isolated autological double-derived cell population enriched with mesenchymal stem cells (MSCS) and methods for obtaining and using the same

Abstract

The present invention relates to an isolated autological double-derived cell population enriched with MSCs and provides detailed methods for obtaining and utilizing these cells for therapeutic purposes. The invention encompasses a unique combination of cells derived from both blood and skin sources, enriched with mesenchymal stem cells (MSCs). The methods described include the isolation of these cells from blood and skin tissues, as well as their subsequent culturing, identification, and administration for therapeutic applications, such as the restoration or enhancement of spermatogenesis and fertility in male subjects. The detailed description encompasses the various steps, procedures, culture conditions, and techniques involved in the acquisition and utilization of the isolated cell population.

Description

[0001] ISOLATED AUTOLOGICAL DOUBLE-DERIVED CELL POPULATION ENRICHED WITH MESENCHYMAL STEM CELLS (MSCS) AND METHODS FOR OBTAINING AND USING THE SAME

[0002] FIELD OF THE INVENTION

[0003] The present invention resides within the field of regenerative medicine and stem cell therapy. It specifically relates to an isolated autological double-derived cell population and provides comprehensive methods for acquiring and employing these cells for therapeutic purposes.

[0004] BACKGROUND OF THE INVENTION

[0005] Conventional stem cell therapies have predominantly focused on the utilization of mesenchymal stem cells (MSCs) derived from a single source, such as bone marrow or adipose tissue. Various origin MSCs have different properties with significant different mechanisms.

[0006] Prior Art US20140308253A1 disclosed an isolated cell, wherein the cell is mitotically competent and expresses Vasa, Oct-4, Dazl, Stella and optionally, an SSEA. In a specific embodiment, the isolated cell is a female germline stem cell and in another specific embodiment, the isolated cell is a female germline stem cell progenitor that expresses SSEA. Preferably, the female germline stem cells, or their progenitor cells, are non-embryonic, mammalian, and even more preferably, human. A method for the isolation of compositions comprising female germline stem cells and / or female germline stem cell progenitors, said method comprising the steps of: a) homogenizing ovarian tissue; b) contacting the tissue with an agent that binds to an SSEA; and c) isolating female germline stem cells and / or female germline stem cell progenitors. The immunomagnetic method was used in biomedical and diagnostic applications to isolate specific target cells or molecules from a complex sample. It involves the use of magnetic beads coated with antibodies or other molecules that can bind to the target of interest.

[0007] Another prior art disclosed by S Saha et. al., Cells. 2021 Jul; 10(7): 1613. (Application of Stem cell Therapy for Infertility) disclosed Mesenchymal Stem Cells (MSCs) are categorized as bone marrow stromal cells, adipose-derived stem cells, menstrual-blood-derived MSCs and umbilical-cord-derived MSCs, amniotic-fluid-derived MSCs, placental-tissue-derived MSCs, salivary-gland-derived MSCs, and dental-pulp-derived MSCs. Various preclinical and clinical studies have reported the potential therapeutic application of MSCs for the treatment of infertility due to ovarian and endometrial dysfunction. MSCs travel to the damaged ovary and settle there to restore ovarian function through the release of various cytokines by paracrine mechanisms. Cytokines such as vascular endothelial growth factor, insulin-like growth factor, and hepatocyte growth factor induce the formation of new vessels, prevent apoptosis as well as fibrosis and thus ameliorate ovarian dysfunction. MSCs also help in the revival of fertility by endometrial regeneration through the release of various bioactive molecules that modulate inflammation and other immune reactions and activate tissue-specific progenitor cells.

[0008] Meanwhile, prior art by YH Cui, et al. Asian J Androl. 2023 Jan-Feb; 25(1): 13-20. (Generation of male germ cells in vitro from the stem cells) provides methods of obtaining functional male gametes for restoring male fertility. Stem cells, including embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), spermatogonial stem cells (SSCs), and mesenchymal stem cells (MSCs), possess the abilities of both self-renewal and differentiation into germ cells. Significantly, much progress has recently been achieved in the generation of male germ cells in vitro from various kinds of stem cells under the specified conditions, e.g., the coculturing with Sertoli cells, three-dimensional culture system, the addition of growth factors and cytokines, and / or the overexpression of germ cell-related genes.

[0009] However, limitations exist in terms of the quantity and regenerative potential of cells obtained from these sources. Therefore, there is a need for alternative cell sources and enrichment methods that can enhance the therapeutic efficacy of stem cell-based treatments such as mixed population of MSCs which provide higher successful rate especially in case of reparative application to the complex multi-stage process.

[0010] SUMMARY OF THE INVENTION

[0011] The present invention addresses the aforementioned need by introducing an isolated autological double-derived cell population. These cells are obtained from both blood and skin sources, combining the advantages of cells derived from two distinct tissues. The isolated cells, enriched with MSCs, provide a valuable resource for various therapeutic applications.

[0012] A method for obtaining blood mononuclear cells enriched with MSCs, comprising: a) administering granulocyte colony-stimulating factor to a patient; b) collecting venous blood from the patient and adding heparin as an anticoagulant; c) separating cells from the blood using a colloidal plasma substitute solution containing modified liquid gelatin; d) resuspending the separated cells in the remaining supernatant; e) culturing the resuspended cells in a culture medium comprising RPMI medium, autologous plasma, and additives; f) fixing the adherent cell fraction obtained after culturing; g) washing the fixed cells and obtaining a cell pellet; h) providing a gradient separation of small-size cells; and i) identifying MSCs within the enriched cultured cells fraction.

[0013] The granulocyte colony-stimulating factor is administered subcutaneously in a dose dependent on the patient's weight and the colloidal plasma substitute solution contains 4% modified liquid gelatin.

[0014] In one aspect of the present invention, the culturing of the resuspended cells includes incubation in a carbon dioxide incubator under standard culture conditions.

[0015] In another aspect of the present invention, the method further comprising adding retinoic acid, bone morphogenetic protein (BMP-4), and Activin A (Act A) to the culture medium during culturing.

[0016] A composition comprising blood mononuclear cells enriched with MSCs obtained according to the method for obtaining blood mononuclear cells enriched with MSCs of the present invention.

[0017] A method for obtaining skin cells enriched with MSCs, comprising: a) collecting a piece of skin tissue from a patient; b) trypsinizing the skin tissue in trypsin-EDTA diluted with saline; c) incubating the resulting solution and biomaterial at 37°C; d) washing the incubated mixture in Hepes-solution; e) placing the washed mixture in culture flasks; f) culturing the skin cells in a culture medium comprising RPMI medium, autologous plasma, and additives; g) providing enrichment of post-culturing cell suspension by MSCs using gradient separation of small-size cells; and h) identifying MSCs within the enriched cultured skin cells fraction. In one aspect of the present invention, the culturing of the skin cells includes incubation in a carbon dioxide incubator under standard culture conditions.

[0018] The method of the present invention comprising adding of retinoic acid, bone morphogenetic protein (BMP-4), and Activin A (Act A) to the culture medium during culturing.

[0019] A composition comprising skin cells enriched with MSCs obtained according to the method for obtaining skin cells enriched with MSCs.

[0020] A method for isolating blood mononuclear cells enriched with MSCs, comprising: a) administering granulocyte colony-stimulating factor to a patient to stimulate cell mobilization; b) collecting venous blood from the patient and adding an anticoagulant; c) separating nucleated cells from the blood using a colloidal plasma substitute solution containing modified liquid gelatin; d) resuspending the separated cells and removing the cell-free supernatant; e) fixing the adherent cell fraction obtained after culturing in a culture medium containing autologous plasma; f) washing the fixed cells to remove non-adherent cells; g) culturing the cells under specific conditions; h) providing a gradient separation by small-size cells; and i) identifying MSCs within cell population.

[0021] In one aspect of the present invention, the separation of nucleated cells is achieved through free accelerated sedimentation of erythrocytes at a temperature of 20-22°C.

[0022] In another aspect of the present invention, the colloidal plasma substitute solution contains a 4% solution of succinylated gelatin.

[0023] The method further comprising counting and assessing the viability of the nucleated cells in the suspension.

[0024] A composition comprising blood mononuclear cells enriched with MSCs obtained according to the method for isolating blood mononuclear cells enriched with MSCs. A method for isolating skin cells enriched with MSCs, comprising: a) collecting a piece of skin tissue from a patient; b) treating the skin tissue with trypsin-EDTA diluted with saline to obtain a cell suspension; c) incubating the cell suspension for a specified period of time at 37°C; d) washing the incubated cell suspension by Hepes-solution; e) placing the washed cell suspension in culture flasks containing a culture medium comprising autologous plasma; f) culturing the skin cells under specific culture conditions; g) providing a gradient separation of small-size cells and h) identifying MSCs within the cultured skin cells.

[0025] In one aspect of the present invention, the culture medium comprises RPMI 1640, autologous plasma, and specific additives.

[0026] The method for isolating skin cells enriched with MSCs, further comprising adding retinoic acid, Bone morphogenetic protein (BMP-4), and Activin A (Act A) to the culture medium during culturing.

[0027] A composition comprising skin cells enriched with MSCs obtained according to the method for isolating skin cells enriched with MSCs

[0028] An isolated autologous double-derived cell population enriched by MSCs obtained from both skin and blood sources for transplantation to a patient.

[0029] The isolated cell population are mammalian cells. The isolated cell population are non- embryonic cells. The isolated cell population are human cells. The isolated cell populations are derived from the patient's own body.

[0030] One of the advantages of the methods of the present invention, by using two types of autologous stem cells from different tissues and preparing them separately can indeed increase the reparative potential. Combining different types of stem cells allows for a synergistic effect, as each type of stem cell has its unique characteristics and abilities. Moreover, by using two different types of autologous stem cells, this will harness the benefits of each type and enhance the overall regenerative capacity.

[0031] To avoid the use of magnetic antibodies in the separation and culturing of autologous stem cells for direct clinical application, multistage methods of the present invention can be employed. This approach involves several steps, including tissue harvesting, initial cell separation, density gradient centrifugation, cell sorting, expansion and culture, and characterization and quality control. By utilizing these stages, it is possible to isolate and cultivate the desired MSCs without relying on magnetic antibodies. It is important to have these methods to the specific stem cell types and therapeutic goals involved for optimal implementation of obtaining such a population from the tissues of adult patient with reproductive problems for further influence on gametogenesis in vitro.

[0032] Further advantages of the system in accordance with the invention and its application can be derived from the description and the accompanying drawings. The above-mentioned features and those to be further described below can be utilized in accordance with the invention individually or collectively in arbitrary combination. The embodiments mentioned are not to be considered exhaustive enumerations, rather have exemplary character. The invention is shown in the drawings and explained more closely with reference to embodiments.

[0033] BRIEF DESCRIPTION OF THE DRAWINGS

[0034] The accompanying drawings, which are incorporated in and constitute a part of the specification, illustrate embodiments of the invention and, together with the description, explain the principles of the invention.

[0035] Figure 1 illustrates human blood cells (adherent fraction) in living not-stained culture. Little cells with big nucleus and dance cytoplasm are well visualized among polymorph cells in accordance of the method of the present invention.

[0036] Figure 2 illustrates human blood cells (adherent fraction) in culture. CD105+ and CD105- cells in accordance of the method of the present invention.

[0037] Figure 3 illustrates skin cells in living not-stained culture with little cells with big nucleus and dance cytoplasm are well visualized at the surface of polygonal cells monolayer in accordance of the method of the present invention.

[0038] Figure 4 illustrates skin cells in culture with CD105+ and CD105- cells in accordance of the method of the present invention. Figure 5 illustrates skin cells in living not-stained culture after retinoic acid addition with little cells with big nucleus and dance cytoplasm are well visualized at the surface of polygonal cells monolayer and sometimes form clusters in accordance of the method of the present invention.

[0039] Figure 6 illustrates skin cells in culture after retinoic acid addition. CD105+ and CD105- cells in accordance of the method of the present invention.

[0040] BRIEF DESCRIPTIONS OF THE INVENTION

[0041] The present invention will now be described in detail in connection with specific embodiments with reference to the accompanying drawings. Unless the context requires otherwise, throughout the specification and claims which follow, the word “comprise” and variations thereof, such as, “comprises” and “comprising” are to be construed in an open, inclusive sense that is as “including, but not limited to”. Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. With respect to the use of substantially any plural and / or singular terms herein, those having skill in the art can translate from the plural to the singular and / or from the singular to the plural as is appropriate to the context and / or application. The various singular / plural permutations may be expressly set forth herein for sake of clarity. It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase "A or B" will be understood to include the possibilities of "A" or "B" or "A and B."

[0042] The term “granulocyte colony-stimulating factor” (G-CSF) such as filgrastim (Neupogen®) or pegfilgrastim (Neulasta®), can be given to be administered to a patient to stimulate the mobilization of cells, particularly MSCs. Method for obtaining blood mononuclear cells with enriched MSCs

[0043] A method for obtaining blood mononuclear cells enriched with MSCs, comprising: a) administering granulocyte colony-stimulating factor to a patient; b) collecting venous blood from the patient and adding heparin as an anticoagulant; c) separating cells from the blood using a colloidal plasma substitute solution containing modified liquid gelatin; d) resuspending the separated cells in the remaining supernatant; e) culturing the resuspended cells in a culture medium comprising RPMI medium, autologous plasma, and additives; f) fixing the adherent cell fraction obtained after culturing; g) washing the fixed cells and obtaining a cell pellet; h) providing a gradient separation of small-size cells; and i) identifying MSCs within the enriched cultured cells fraction.

[0044] In one aspect of the present invention, the Granulocyte colony-stimulating factor is administered subcutaneously in a dose dependent on the patient's weight and the colloidal plasma substitute solution contains 4% modified liquid gelatin. The purpose of accelerated "spontaneous" sedimentation of erythrocytes of a colloidal plasma substitute solution containing 4% modified liquid gelatin (and which is approved by the Ministry of Health of Ukraine for intravenous use), as well as a change in the temperature regime i.e. sedimentation of erythrocytes is carried out at a temperature of 20-22° C for 45-60 minutes. For concentration, a careful centrifugation procedure is used to reduce the volume of the sample having the nucleated cells after removing the majority of erythrocytes. A change of the condition of adhesion is provided to the way of minimal using foreign biological material. In the present invention, adhesion of blood MSCs is provided to the plastic surface. In addition, the adhesion time is reduced to 8 hours trying to get the fraction of more active MSCs.

[0045] Meanwhile, culturing of the resuspended cells includes incubation in a carbon dioxide incubator under standard culture conditions with adhesive fraction cultured in vitro during 3 days. During culturing, retinoic acid, bone morphogenetic protein (BMP-4), and Activin A (Act A) are added to the culture medium. Such duration (cultured in vitro during 3 days) is comparable with cell cycle time of most active stem cells and that is why it should be enough for stabilization of cell population and differentiation way direction which is carried out by addition of retinoic acid, BMP-4 and Activin A. The reduction of duration in vitro without control of immune system and absence of culture passaging of cell population promotes gene stability of autologous cell fraction.

[0046] Another important point of the present invention is that the clinical cell application is provided with animal-free culture medium for cell culturing. In one aspect of the present invention, plasma of an infertile patient is obtained at the time of blood sampling. This is to avoid the contact with non-detectable pathogens and inducer of undesirable changes in personal human cell population. Culture condition contributes to implementation of different cells fate program. MSC cells stay small, ready to proliferation and migration unlike other types of cells in population. For separation the fraction of small size cells (7-9 pm), double density gradient of iohexol solutions is used for blood cells separation. A composition comprising blood mononuclear cells enriched with MSCs obtained according to the method of the present invention.

[0047] One aspect of the invention involves a detailed method for obtaining blood mononuclear cells with enriched MSCs. The method comprises the following steps

[0048] 1. Administration of Granulocyte colony-stimulating factor:

[0049] • Granulocyte colony-stimulating factor, is administered to a patient to stimulate the mobilization of cells, particularly MSCs.

[0050] 2. Collection and Anticoagulation of Venous Blood:

[0051] • Venous blood is collected from the patient using standard techniques, and an anticoagulant, such as heparin, is added to prevent clotting.

[0052] 3. Separation of Cells Using Colloidal Plasma Substitute Solution:

[0053] • The collected blood is subjected to cell separation using a colloidal plasma substitute solution containing modified liquid gelatin. This solution facilitates the isolation of MSCs.

[0054] 4. Resuspension and Culture of Separated Cells:

[0055] • The separated cells are resuspended in the remaining supernatant and cultured in a specific culture medium comprising RPMI, autologous plasma, and additives. Non-adherent fraction of cells was removed. This culture step promotes the adherence and proliferation of MSCs.

[0056] 5. Enrichment of post-culturing cell suspension by MSCs

[0057] • After the end of culturing the adherent cells were removed and undergo gradient separation for small-size cells selection. This step allows to avoid antibody binding and stay suitable for further clinical application.

[0058] 6. Cell Fraction Enriched by MSCs expressing CD105+:

[0059] Small aliquot of cell suspension was taken for immunohistochemistry visualization and counting of CD105+. • The isolated cell fraction enriched by MSCs expressing CD105+ can be administered to a target tissue for therapeutic purposes, such as transplantation to restore or enhance cellular function.

[0060] Examples

[0061] Prior to blood sampling, the patient is prescribed a Granulocyte colony-stimulating factor (30- 60 Mill-dose depends of patient weight) was injected subcutaneously. After 24 hours taking the Granulocyte colony-stimulating factor, venous blood (200 ml) was collected from the patient. Heparin (2500 III per 100 ml of blood) was added as anticoagulant. About 20 ml of blood was used for autological plasma was obtained. The preparation of the cell suspension according to a prototype [Harris D. T., Schumacher M. J., Rychlik S. et al. Collection, separation, and cryopreservation of umbilical cord blood for use in transplantation / / Bone Marrow Transplant. - 1994. - B13. - P. 135-143] and according to the method was carried out in parallel on the same samples of biomaterial. Biological material for research was taken in the same quantity. When carrying out the separation of cells according to the prototype, a solution was used, which contained gelatin in a concentration of 3% (the composition of the solution also included glucose, acidic sodium citrate, bi-distilled water). After preparation of the separation of the cells, the solution was poured into vials with a capacity of 50 ml, which were sealed and sterilized in the autoclave mode (1.2 atmospheres for 30 minutes). Mixing was carried out gradually by layering gelatin solution on native blood in a 1 :1 ratio. After careful mixing of the gelatin solution by shaking and ensuring thorough distribution of the gelatin solution within the blood, the gelatin solution on native blood was left for free accelerated sedimentation of erythrocytes at a temperature of +4°C on a tripod in a refrigerator chamber for 45, 55, 60 minutes. Then, the supernatant was transferred to a special container using a plasma extractor and centrifuged at 1200 rpm for 8 minutes. After that, the cell-free plasma was carefully removed, and the cell sediment was resuspended in the remaining plasma. Thereafter, the cells in the suspension were counted, their viability was studied by the method of supravital staining, and the cell composition of the suspension in smears was also studied. When performing the examples according to the above method, a colloidal plasma substitutes solution containing 4% of modified liquid gelatin was carefully added to the blood container in a ratio of 1 : 1 , after which exposures were made for 45, 55, 60 minutes (the duration of the settling procedure depended on the characteristics of the specific sample) at a temperature of 20-22 °C. Blood leukoconcentrate was concentrated by gentle centrifugation at 1200 rpm for 8 minutes at room temperature. The cell-free supernatant was carefully removed, and the cells were resuspended in the remaining supernatant. It was found that the percentage of nucleated cells isolated by the prototype is lower than when isolated by the method of the present invention. In addition, when using the prototype method, the loss of nucleated cells in general is much greater and this can be observed on the quality of the samples. The results indicate that the sedimentation of erythrocytes according to the prototype is much slower, and thus the sedimentation procedure is prolonged. At the same time, there are conditions that prevent the free sedimentation of erythrocytes i.e. the gelatin solution forms a gel-like structure when the temperature drops, which contributes both to the retention of erythrocytes and to the "mixing" of nucleated cells, which have a high density with erythrocytes. In addition, at a temperature close to zero, nucleated cells are sensitized to low temperature conditions, which additionally causes a decrease in their preservation during further freezing and thawing. The results of bacteriological control show that the samples prepared according to the prototype have a significantly higher percentage of microflora contamination than the samples prepared according to the method of the present invention. This indicates an increase in the probability of a violation of sterility, which may be related to the need to prepare a 3% gelatin solution, as well as the need to bring containers with the material into a refrigerator for precipitation at a temperature of +4°C. The method of the present invention has better results in the preservation of nucleated cells, which includes stem cells. At the same time, the use of a 4% solution of succinylated gelatin significantly simplifies and shortens the procedure for the selection of nucleated cells. All stages of the procedure are such as to protect the cells from severe mechanical damage.

[0062] Due to single-use materials that have permission for clinical use, as sterile, non-toxic, the method of the present invention meets the conditions of sterility in working with blood and other human biological material. The resulting cell pellet was examined with a light microscope and the total number of cells and the percentage of living were counted using a Makler’s chamber after staining with trypan blue. Thereafter, the cells were resuspended in culture medium, which consist of RPMI, 20% autological plasma of patient, then transferred to culture flasks and kept for 8 hours in a carbon dioxide (CO2) incubator under standard culture conditions (t - 370C, 5% CO2) to fix the adherent cell fraction. At the end of the experiment, the non-adherent cells fraction was removed, and the fixed layer of cells was washed 3 times with physiological saline.

[0063] After that cell pellet was culturing in the medium, which consists of RPMI (growth medium used in cell culture), 20% autological plasma of patient, 1% GlutaMax Supplement, 1% Antibiotic-Antimycotic, in a CO2 incubator under standard culture conditions (t - 370C, 5% CO2) during 48 hours. After that 1 pM retinoic acid, 20 ng / ml BMP-4, Activin A 100 ng / ml were added to the culture medium and culturing 24 hours. Visual control and photography were carried out with using of inverted microscope. During the 12-24 hours of cultivation, the adherent fraction of blood mononuclear cells becomes polymorphic. It was observed that cells of different shapes and sizes - round, spindle-shaped, triangular, with processes and without. Some of these cells acquire a very large size and form a vacuolated cytoplasm and a small asymmetric nucleus as illustrated in Figure 1. MSCs CD105 positive cells were stained using monoclonal antibody and secondary antibody according with manual protocol. Visualization of positive cells was made by Multivision Polymer Detection. Cell nuclei were stained by hematoxylin. After that Percentage of CD105+ were calculated in the tan fields of view in each sample. Among these cells, there are rounded small cells with dense cytoplasm, a large nucleus and the absence of processes. According to immunohistochemistry, they are CD105+ MSCs as shown in Figure 1 and Figure 2. The number of such cells in the experiment was 16 ± 1.7% of all nuclear cells in the population. After gradient separation the cell population enriched by MSCs was obtained. The number of CD105+ consisted 35 ± 2.1 %.

[0064] Obtaining of autoloqical cells suspension derived from blood mononuclear enriched by MSCs and committed for spermatogenesis

[0065] The research study group included five male patients with an average age of 27- 39 years old. Exclusion criteria of the patients were chronic disease and a BMI higher than 25. Prior to 24 hours before blood sampling patient underwent the Neupogen stimulation (60MILI). His venous blood (200 ml) collected, and heparin (2500 III per 100 ml of blood) was added as an anticoagulant. The fraction of nucleated cells isolated using gelofusine, a succinylated gelatin solution. It is a colloid solution that is commonly used as a volume expander or plasma substitute in medical settings. Then washed twice in the Hank's balanced salt solution (HBSS). The resulting cell pellet examined with a light microscope, the total number of cells and the percentage of living counted. The living mononuclear accounted 97% of mononuclear suspension. Thereafter, the cells resuspended in culture medium, which consist of RPMI, 20% autological plasma of patient, 1 % GlutaMax Supplement, 1 % Antibiotic-Antimycotic, in a carbon dioxide (CO2) incubator under standard culture conditions (temperature - 370C, 5% CO2) during 48 hours. Non-adherent cells fraction was removed after first 8 hours during this period. After that 1 pM RA, 20 ng / ml BMP-4, Activin A 100 ng / ml were added to the culture medium and culturing next 24 hours. Then cells were removed mechanically and separated in the density gradient of iohexol solutions. Mononuclear cells suspension diluted with HBSS is layered on double density gradient of iohexol solutions (upper layer is 1080 kg / m3, and lower - 1090 kg / m3). After centrifugation at 700g, a ring containing cells 7-9 pm appears on the boundary between these gradients. These cells were collected and washed twice by physiological saline. Living cells accounted 91 ±1,2 %, CD105+ cells - 35±2,1% of this suspension.

[0066] Method for Obtaining Skin Cells Enriched with MSCs

[0067] A method for obtaining skin cells enriched with MSCs, comprising: a) collecting a piece of skin tissue from a patient; b) trypsinizing the skin tissue in trypsin-EDTA diluted with saline; c) incubating the resulting solution and biomaterial at 37°C; d) washing the incubated mixture in Hepes-solution; e) placing the washed mixture in culture flasks; f) culturing the skin cells in a culture medium comprising RPMI medium, autologous plasma, and additives; g) providing enrichment of post-culturing cell suspension by MSCs using gradient separation of small-size cells; and h) identifying MSCs within the enriched cultured skin cells fraction.

[0068] In one aspect of the present invention, the culturing of the skin cells includes incubation in a carbon dioxide incubator under standard culture conditions. During culturing, retinoic acid, bone morphogenetic protein (BMP-4), and Activin A (Act A) are added to the culture medium. Suspension human skin cells without feeder layer is grown, using autologous patient plasma and avoiding long-term culture or passaging in 3-day duration in vitro is comparable with cell cycle time of most active stem cells. Other trophic factors such as retinoic acid, Brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) are provided to directly stimulate cultured skin cells. At a final day (the 3rdday) the BMP-4 and Activin A are added in culture medium for germ way differentiation direction. MSC cells stay small, ready to proliferation and migration unlike other types of cells in population. For separation the fraction of small size cells (7-9 pm), double density gradient of iohexol solutions is used for blood cells separation. A composition comprising skin cells enriched with MSCs obtained according to the method of the present invention.

[0069] Another aspect of the invention involves a detailed method for obtaining skin cells enriched with MSCs. The method comprises the following steps:

[0070] 1. Collection of Skin Tissue:

[0071] • A piece of skin tissue is collected from the patient using standard surgical techniques. 2. Trypsinization and Incubation:

[0072] • The collected skin tissue is treated with trypsin-EDTA diluted with saline to obtain a cell solution. The cell solution is then incubated at 37°C for a specified period of time.

[0073] 3. Washing and Plating of Cell Solution:

[0074] • The incubated cell solution is washed in Hepes-solution and placed in culture flasks containing a culture medium comprising autologous plasma. This allows for the adherence and growth of MSCs.

[0075] 4. Culture and Identification of MSCs:

[0076] • The skin cells are cultured under specific conditions, including the use of a culture medium comprising RPMI 1640, autologous plasma, and specific additives. MSCs within the cultured skin cells are identified using appropriate techniques, such as immunocytochemistry.

[0077] 5. Enrichment of post-culturing cell suspension by MSCs

[0078] • The removed cells undergo gradient separation for small-size cells selection. This step allows to avoid antibody binding and stay suitable for further clinical application.

[0079] Examples

[0080] The piece of skin tissue (size 10 x 5 x 8 mm) was taken with special scissors and syringe needles. The obtained skin tissue as biomaterial was trypsinized in trypsin-EDTA (0.25%), which was diluted with saline concentration of 1 : 1. Thereafter, the resulting solution together with the biomaterial was put on display for 1 hour at 37°C. Next, the resulting mixture was washed in Hepes-solution and placed into culture flacons. Skin cells were incubated in carbon dioxide (CO2) incubator under standard culture conditions (temperature - 370C, 5% carbon dioxide (CO2)) during 48 hours. Culture medium consists of RPMI 1640, 20% autological plasma of patient, 1% GlutaMax Supplement, 1% Antibiotic-Antimycotic, 40 ng / ml BDNF, 80 ng / ml NGF, 1 pM retinoic acid. After that 1 pM retinoic acid, 20 ng / ml BMP-4, Activin A 100 ng / ml were added to the culture medium and culturing 24 hours. Visual control and photography were carried out with using of inverted microscope. After period of culturing, cell material was removed using trypsin. At the early period of culturing (12-24hours) it was observed adhesion of skin cells of two main phenotypic appearance (some cells had big elongated nucleus with several nucleoli, some cells had one large round nucleus) at the stages of settling and fixation to the plastic surface as shown in Figure 3. The adhesion of skin cells of different phenotypic begins to form a monolayer, which consists of polygonal cells of the same type that are closely adjacent to each other. MSC CD105 positive cells was stained in small aliquot of cells suspension using monoclonal antibody and secondary antibody according with manual protocol. Visualization of positive cells was made by Multivision Polymer Detection. Cell nuclei were stained by hematoxylin. After that Percentage of CD105+ were calculated in the tan fields of view in each sample. Among them there are single small rounded cells with dense cytoplasm and a large nucleus, which are CD 105 positive according to histochemical staining as illustrated in Figure 3 and Figure 4. The number of such cells 18.0 ± 2.5% of all living cells in the population. In this case, it was observed the formation of cell clusters, which consisted of 5-20 small rounded cells of the same type as depicted in Figure 5 and Figure 6. After gradient separation, the cell population enriched by MSC was obtained. The number of CD105+ cells consisted 41 ± 3.2%. Mononuclear cells suspension was diluted with Hank’s solution and was layered on double density gradient of iohexol solutions. Density gradient of the upper layer is 1080 kg / m3, and lower - 1090 kg / m3. After centrifugation at 700 g force, a ring containing cells 7-9 pm appears on the boundary between these gradients. This method provides reduced contamination of the released cells 7-9 pm size with other cell types while maintaining a sufficient portion of viable cells.

[0081] Obtaining of autological cells suspension derived from skin enriched by MSCs and committed for spermatogenesis

[0082] Few pieces of skin tissues were taken with special scissors and syringe needles. The obtained material trypsinized in trypsin-EDTA (0.25%), which diluted with saline 1 : 1 , after of which the resulting solution together with the biomaterial put on displayed for 1 hour at 37° C. Then, the resulting mixture was washed in Hepes-solution and placed into culture flacons. Culture medium consisted of RPMI, 20% autological plasma of patient, 1% GlutaMax Supplement, retinoic acid 1 pM, 80 ng / ml NGF (nerve growth factor) and 40 ng / ml BDNF (brain-derived neurotrophic factor), 1% Antibiotic-Antimycotic, in a CO2 incubator under standard culture conditions (temperature - 370C, 5% CO2) during 48 hours. Non-adherent cells fraction was removed after first 8 hours during this period. After that 20 ng / ml BMP-4, Activin A 100 ng / ml were added to the culture medium and culturing next 24 hours. Then cells were removed using trypsin (0.25%), which was diluted with culture medium 1 : 1 , after which the resulting mixture together with the cell complexes were placed for 10 minutes of exposure to the incubator (temperature = 37 C) followed by pipetting. After that cell suspension was rinsed by Hank's balanced salt solution (HBSS) 3 times by centrifugation and separated in the density gradient of iohexol solutions. Mononuclear cells suspension diluted with Hank's balanced salt solution (HBSS) was layered on double density gradient of iohexol solutions (upper layer is 1080 kg / m3, and lower - 1090 kg / m3). After centrifugation at centrifugation at 700 g force, a ring containing cells 7-9 pm appears on the boundary between these gradients. These cells were collected and washed twice by physiological saline. Living cells accounted 85%, CD105+ cells - 40% of this suspension.

Claims

CLAIMS1. A method for obtaining blood mononuclear cells enriched with mesenchymal stem cells (MSCs), comprising: a) administering granulocyte colony-stimulating factor to a patient; b) collecting venous blood from the patient and adding heparin as an anticoagulant; c) separating cells from the blood using a colloidal plasma substitute solution containing modified liquid gelatin; d) resuspending the separated cells in the remaining supernatant; e) culturing the resuspended cells in a culture medium comprising RPMI medium, autologous plasma, and additives; f) fixing the adherent cell fraction obtained after culturing; g) washing the fixed cells and obtaining a cell pellet; h) providing a gradient separation of small-size cells; and i) identifying MSCs within the enriched cultured cells fraction.

2. The method of claim 1 , wherein the granulocyte colony-stimulating factor is administered subcutaneously in a dose dependent on the patient's weight.

3. The method of claim 1 , wherein the colloidal plasma substitute solution contains 4% modified liquid gelatin.

4. The method of claim 1 , wherein the culturing of the resuspended cells includes incubation in a carbon dioxide incubator under standard culture conditions.

5. The method of claim 1 , further comprising adding retinoic acid, bone morphogenetic protein (BMP-4), and Activin A (Act A) to the culture medium during culturing.

6. A composition comprising blood mononuclear cells enriched with MSCs obtained according to the method of claim 1.

7. A method for obtaining skin cells enriched with mesenchymal stem cells (MSCs), comprising: a) collecting a piece of skin tissue from a patient; b) trypsinizing the skin tissue in trypsin-EDTA diluted with saline; c) incubating the resulting solution and biomaterial at 37°C;d) washing the incubated mixture in Hepes-solution; e) placing the washed mixture in culture flasks; f) culturing the skin cells in a culture medium comprising RPMI medium, autologous plasma, and additives; g) providing enrichment of post-culturing cell suspension by MSCs using the gradient separation of small-size cells; and h) identifying MSCs within the enriched cultured skin cells fraction.

8. The method of claim 7, wherein the culturing of the skin cells includes incubation in a carbon dioxide incubator under standard culture conditions.

9. The method of claim 7, further comprising adding of retinoic acid, bone morphogenetic protein (BMP-4), and Activin A (Act A) to the culture medium during culturing.

10. A composition comprising skin cells enriched with MSCs obtained according to the method of claim 7.

11. A method for isolating blood mononuclear cells enriched with mesenchymal stem cells (MSCs), comprising: a) administering granulocyte colony-stimulating factor to a patient to stimulate cell mobilization; b) collecting venous blood from the patient and adding an anticoagulant; c) separating nucleated cells from the blood using a colloidal plasma substitute solution containing modified liquid gelatin; d) resuspending the separated cells and removing the cell-free supernatant; e) fixing the adherent cell fraction obtained after culturing in a culture medium containing autologous plasma; f) washing the fixed cells to remove non-adherent cells; g) culturing the cells under specific conditions; h) providing a gradient separation by small-size cells; and i) identifying MSCs within cell population.

12. The method of claim 11 , wherein the separation of nucleated cells is achieved through free accelerated sedimentation of erythrocytes at a temperature of 20-22°C.

13. The method of claim 11 , wherein the colloidal plasma substitute solution contains a 4% solution of succinylated gelatin.

14. The method of claim 11 , further comprising counting and assessing the viability of the nucleated cells in the suspension.

15. A composition comprising blood mononuclear cells enriched with MSCs obtained according to the method of claim 11.

16. A method for isolating skin cells enriched with mesenchymal stem cells (MSCs), comprising: a) collecting a piece of skin tissue from a patient; b) treating the skin tissue with trypsin-EDTA diluted with saline to obtain a cell suspension; c) incubating the cell suspension for a specified period of time at 37°C; d) washing the incubated cell suspension by Hepes-solution; e) placing the washed cell suspension in culture flasks containing a culture medium comprising autologous plasma; f) culturing the skin cells under specific culture conditions; g) providing a gradient separation of small-size cells and h) identifying MSCs within the cultured skin cells.

17. The method of claim 16, wherein the culture medium comprises RPMI 1640, autologous plasma, and specific additives.

18. The method of claim 16, further comprising adding retinoic acid, Bone morphogenetic protein (BMP-4), and Activin A (Act A) to the culture medium during culturing.

19. A composition comprising skin cells enriched with MSCs obtained according to the method of claim 16.

20. An isolated autologous double-derived cell population enriched by MSCs obtained from both skin and blood sources for transplantation to a patient.

21. The isolated cell population of claim 1 , wherein the cells are mammalian cells.

22. The isolated cell population of claim 1 , wherein the cells are non-embryonic cells.

23. The isolated cell population of claim 1, wherein the cells are human cells.

24. The isolated cell population of claim 1 , wherein the cells are derived from the patient's own body.

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