Lyophilized powder enriched with growth factors

Abstract

The invention of product and method of producing lyophilized powder enriched with growth factors using conditioned medium produced with the help of growth medium supplement based on umbilical cord mesenchymal stem cells for the production of growth stimulants and tissue repair in the human body is a method of producing the product which is a kind of lyophilized powder enriched with growth factors using conditional medium produced with the help of growth medium supplement based on the umbilical cord mesenchymal stem cells to produce the growth stimulants and repairing tissue in the body. The present invention also related to the product of lyophilized powder with long-term durability and a method to enrich lyophilized powder extracted from umbilical cord mesenchymal stem cells and the growth stimulants produced from enriched lyophilized powder for mesotherapy as well as tissue and wound repair.

Description

[0001] TITLE OF INVENTION

[0002] LYOPHILIZED POWDER ENRICHED WITH GROWTH FACTORS

[0003] TECHNICAL FIELD OF THE INVENTION

[0004] The present invention is related to the production method of lyophilized powder enriched with growth factors extracted from the process of stem cell propagation and also related to the final product of lyophilized powder and also related to the product of lyophilized powder with long-term durability and also related to a method to enrich lyophilized powder extracted from umbilical cord mesenchymal stem cells and also related to the growth stimulants produced from enriched lyophilized powder for mesotherapy as well as tissue and wound repair.

[0005] PRIOR ARTS

[0006] The term stem cell describes a cell with high potential in carrying out multiple cell divisions, which has a unique ability to produce cells similar to itself, and through the processes of self-renewal and distinction, it is able to transform into different types of cells, for the initial construction of tissues and the repair of damaged body tissues. The stem cells are divided to different parts according to their origin. The embryonic stem cells which are obtained from the Inner cell mass in the blastocyst stage. Blastocyst is one of the embryonic stages which morphologically resemble a hollow ball. This ball is trophoblastic which made the placenta. A number of cells are gathered inside this ball, which will turn into an embryo in the next stages. If this mass of internal cells is removed and cultured in a laboratory environment, embryonic stem cells are created. The mature stem cells which are the undifferentiated cells which are existed in whole the newborns and mature. These cells have the ability to renew and differentiate into the main types of specific cells of the tissue, by dividing they fill the place of dead cells and repair damaged tissues. Tissues with mature stem cells are: bone marrow, fat tissue, dental pulp, peripheral blood, brain, spinal cord, blood vessels, skeletal muscle, cornea, retina, pancreas, liver, digestive system. Also the extra-embryonic stem cells which include amnion epithelial stem cells, placental stem cells, amniotic fluid stem cells, and umbilical cord stem cells. Umbilical cord stem cells include umbilical cord blood stem cells, umbilical vein sub endothelial stem cells, umbilical cord matrix stem cells (Wharton's jelly).

[0007] Mesenchymal stem cells are isolated from three relatively distinct compartments on Wharton jelly that can isolate them from peri vascular, inter vascular and also sub amniotic area. It seems that blood stem cells and umbilical cord matrix are intermediate between adult and embryonic cells.

[0008] Wharton's jelly of the umbilical cord contains connective tissue derived from extraembryonic mesoderm and fibroblast-like cells. These cell populations are called umbilical cord mesenchymal stem cells, which have the ability to multiply indefinitely and differentiate into different tissues. Evidence shows that human umbilical cord stromal mesenchymal stem cells have more embryonic characteristics than adult stem cells. The umbilical cord contains two umbilical cord arteries and a umbilical cord vein, and both are located in a special mucous connective tissue called Wharton's jelly, which is covered by the amniotic epithelium. The umbilical cord is considered a hospital waste and the collection of umbilical cord mesenchymal stem cells is non-invasive, in addition, access to umbilical cord mesenchymal stem cells is not cumbersome and has no ethical problems. Umbilical cord mesenchymal stem cells, similar to mesenchymal stem cells extracted from other sources, have a distinct capacity for self-renewal, while maintaining their pluripotency and having the ability to differentiate into adipocytes, osteocytes, chondrocytes, neurons, and hepatocytes. In addition, umbilical cord mesenchymal stem cells have received a lot of attention due to their immune modulating properties. Compared to bone marrow mesenchymal stem cells and embryonic stem cells, umbilical cord mesenchymal stem cells are more similar to fetal stem cells in terms of gene expression (differentiation) and show faster self-renewal than bone marrow mesenchymal stem cells.

[0009] In this regard, the previous researches as well as the registered inventions that have been presented to some extent in the mentioned field are presented below. Among these patent can mention to an Australian invention with patent number AU2016203692B2 which is granted dated 03 / 01 / 2019 titled “Metabolized conditioned growth medium and methods of use” comprising metabolized conditioned growth medium and / or metabolized cell extract and methods of use are described. The metabolized conditioned growth medium and metabolized cell extract compositions may be formulated with an acceptable carrier into injectable or topical formulations, for example, as a cream, lotion or gel, and may be used in cosmeceutical or pharmaceutical applications. The metabolized conditioned growth medium and metabolized cell extract may also be further processed to concentrate or reduce one or more factors or components contained within the metabolized conditioned growth medium or metabolized cell extract. The growth medium may be conditioned by any eukaryotic cell. The metabolized conditioned growth medium and metabolized cell extract may be used to prevent or treat a condition, for example, a skin condition.

[0010] A Chinese invention with patent number CN 103251649 A which is granted dated 19 / 10 / 2018 titled “Lyophilized powder of human mesenchymal stem cell culture supernatant and preparation method thereof’ relates to lyophilized powder of human mesenchymal stem cell culture supernatant and a preparation method of the lyophilized powder. The preparation method comprises the following steps of: culturing a sub-culturing human mesenchymal stem cell by using a mesenchymal stem cell culture medium containing autologous serums; collecting a cell culture supernatant with low sub-culturing times; and preparing the collected supernatant into the lyophilized powder. The lyophilized powder prepared by the preparation method of the lyophilized powder of the human mesenchymal stem cell culture supernatant effectively preserves various cell factor mixtures with biological activities in the human mesenchymal stem cell culture supernatant, is capable of effectively solving the problem of short storage life of the supernatant and lays a foundation for an industrial application of the human mesenchymal stem cell culture supernatant.

[0011] Another Chinese invention with patent number CN104027794B which is granted dated 15 / 06 / 2016 titled “Human umbilical cord mesenchymal stem cells multiple cytokine repairs the application in the biological preparation of skin injury in preparation” relates to a kind of human umbilical cord mesenchymal stem cells multiple cytokine and repair the application in the biological preparation of skin injury in preparation, belong to pharmaceutical technology field which prepared by following steps and obtained: 1) from human umbilical tissue, umbilical cord mesenchymal stem cells is extracted; 2) umbilical cord mesenchymal stem cells is carried out in vitro amplification cultivation; 3) umbilical cord mesenchymal stem cells is used the conditioned medium of serum-free instead and is cultivated, and culture medium is collected; 4) conditioned medium is centrifuged purification, the material of molecular cut off 3.5kDa; 5) by centrifugal wild Oryza species (containing>3.5kDa molecule) carry out dialysis concentration, obtain concentrated solution; 6) concentrated solution is carried out frozen dried, obtain human umbilical cord mesenchymal stem cells multiple cytokine lyophilized powder.

[0012] And a Chinese invention with publication number CN111378616A which is filed dated 14 / 01 / 2020 titled “Research method for repairing skin by adopting prepared stem cell freeze-dried powder” discloses a method for repairing and researching skin by preparing stem cell freeze-dried powder, which comprises the steps of extracting mesenchymal stem cells, cultivating the mesenchymal stem cells, preparing the stem cell freeze-dried powder, performing skin wound healing experiments and performing data statistical analysis. The invention has the beneficial effects that: when the mesenchymal stem cells are extracted, the liquid to be separated in the liquid storage tank is sucked out by the delivery pump through the inlet pipe, the required MSC liquid is left after being filtered by the filter screen and flows out to the culture medium, and the culture sleeve is provided with a plurality of cavities which can be used for accommodating the culture medium, so that accidents such as infection in the transportation process are avoided, and the problem of mutual infection is also avoided; the oxygen concentration of the mesenchymal stem cell culture is 2-3%, the temperature is 37C, the concentration of the seed and carbon dioxide is 5%, and under the condition of low oxygen concentration, the expansion of the adult stem cells is promoted, and the cell doubling period is shortened; the lyophilized powder of the adult stem cells contains polypeptide substances such as epidermal growth factors, fibroblast growth factors and the like, and can repair damaged cells and promote the growth and proliferation of skin cells.

[0013] Also a Japanese invention with patent number JP7090026B2 which is granted dated 23 / 06 / 2022 titled “Adipose tissue-derived mesenchymal stromal cell conditioned medium and method for preparing and using it” generally relates to a composition containing stem cell secretions derived from a conditioned medium, and a method for producing and using the same. Also provided method is a pharmaceutical composition comprising an effective amount of any of the lyophilized compositions described herein and a sustained release drug delivery matrix. For example, a sustained release drug delivery matrix is biodegradable and I or biocompatible. The method for producing the freeze-dried composition is as following: a) enzymatic digestion of adipose tissue to obtain a population of adipocytes, wherein the population of adipocytes comprises at least one adipose stem cell (ASC). b) Adipocytes are cultured in a first culture medium at a seeding density of 2-4 x 10 5 cells I cm 2. c) Passing the cells at least once in the first culture medium, d) Selecting cells, wherein at least 90% of the selected cells express one or more pericyte markers, e) The selected cells are cultured in a second culture medium, wherein the second culture medium is serum-free and contains 20 ng / ml TNFa or 10 ng / ml IFNy. f) Transfer the selected cells into a basal culture medium containing no inflammatory cytokines, g) from the basal culture medium to produce a cell-free conditioned medium containing the secretome of the fat cells, h) lyophilizing the conditioned medium.

[0014] Also an US invention with patent number US10683482B2 which is granted dated 16 / 06 / 2020 titled “Stem cell conditioned media and methods of using the same” is methods of producing stem cell conditioned media to treat mammalian injuries or insults. In at least one embodiment of a method of producing a stem cell conditioned media of the present disclosure, the method comprises the steps of culturing at least one stem cell in a first cell culture medium, replacing some or all of the first cell culture medium with a second cell culture medium and further culturing the at least one stem cell in the second cell culture medium, and collecting a quantity of the second cell culture medium after a culture duration, wherein the quantity of the second cell culture medium contains a cell culture byproduct effective to treat a mammalian insult or injury. In an additional embodiment, the method further comprises the step of processing the quantity of the second cell culture medium using a process selected from the group consisting of centrifugation, concentration, fractionation, lyophilization, and freeze-drying, to create a processed product.

[0015] Another Chinese invention with patent number CN104487568B which is granted dated 15 / 08 / 2017 titled “The mesenchymal stem cells of derived from human embryonic stem, method and its application” is generally relates to mesenchymal stem cells "hES T MiSC" or "T MSC" and the method for producing the stem cell. Methods described is included in CMC model embryonic stem cell as described below: Embryonic stem cell breaks up and developed by intermediate product trophocyte, and cultivates the trophocyte of the differentiation and then break up to hES T MSC or T MSC. Also illustrate cell derived from T MSC or cell line “T MSC DL”. The methods described includes: (a) hESCs or iPSCs2-5 days are cultivated in the culture medium comprising bone morphogenetic protein 4 (BMP4), it is sufficient to make hESCs Or iPSCs is divided into trophoderm. (b) trophocyte is dispersed into unicellular, (c) by the trophocyte be layered on again coating gelatin, vitronectin, laminin, fibronectin, matrigel or On the plate of collagen, (d) cultivated in mescenchymal stem cell (MSC) growth medium comprising the 20%FBS and a-MEM of nonessential amino acid Trophocyte 4-10 days, the amount of the culture medium induces the trophocyte to be divided into mescenchymal stem cell enough, Mescenchymal stem cell described in wherein at least 90% expresses CD73.

[0016] Another Chinese invention with publication number CN103013911A which is filed dated 30 / 11 / 2012 titled “Method for culturing human umbilical cord mesenchymal stem cells through combination of adherence density gradient method and EGF (Epidermal Growth Factor)” provides a method for culturing human umbilical cord mesenchymal stem cells through combination of an adherence density gradient method and as EGF (Epidermal Growth Factor). The method comprises the following steps of: seperation of the UC-MSCs (Umbilical Cord Mesenchymal Stem Cells), primary culture of the UC-MSCs, subculture and augmentation of the UC-MSCs, cryopreservation and resuscitation of the UC-MSCs and detection of UC-MSCs immunophenotyping. According to the method, 4ng / ml of EGF is added by adopting the adherence density gradient method, adherent culture and digestion time control are combined, so that the UC-MSCs with higher purity and activity is obtained; and the method combining the adherence density gradient method and the EGF has the advantages of simple operation, rapidness, practicability and the like and is an effective method for separating and purifying the UC-MSCs.

[0017] Also a Chinese invention with patent number CN105586309B which is granted dated 25 / 01 / 2019 titled “A method of obtaining safe and effective umbilical cord mesenchymal stem cells” provides a kind of method for obtaining safe and effective umbilical cord mesenchymal stem cells, it takes the methods of to detect investigation, add antibiotic in umbilical cord acquisition, umbilical cord transport, umbilical cord preparation and cell cultivation process and guarantees to obtain free of contamination umbilical cord mesenchymal stem cells to guarantee safety, separative efficiency is improved using the cultivating system and cultural method of improvement in separation, incubation, shorten cultivation cycle and the excellent cell of high-purity, characterization can be obtained.The present invention not only can be effectively reduced cost but also can be quickly obtained good mescenchymal stem cell, reduce the injury to cell, shorten cultivation cycle, reduce pollution, and can be improved cell purity.

[0018] A Chinese invention with patent number CN106474155B which is granted dated 06 / 08 / 2019 titled “External-use gel preparation containing human umbilical cord mesenchymal stem cells extract and its preparation method and application” discloses a kind of external-use gel preparation and its preparation method and application containing human umbilical cord mesenchymal stem cells extract, contain human umbilical cord mesenchymal stem cells extract and gel solution, wherein human umbilical cord mesenchymal stem cells extract is the extract after hungry culture, gel solution contains sodium alginate and Multiple electrolytes injection, and contain moisturizer, skin penetration enhancer, form it into the spawn of stable uniform.lt can be used for preparing the drug of the diseases such as treatment skin ulcer, burn, scald, eczema, wound.

[0019] Another Chinese invention with publication number CN 112111450A which is filed dated 02 / 11 / 2020 titled “Culture method and application of umbilical cord mesenchymal stem cells” discloses a culture method and application of umbilical cord mesenchymal stem cells, wherein the method comprises the following steps: compared with the prior art, the method has the advantages that the cell preparation, the cell culture and the cell cryopreservation can avoid the mutual pollution of pathogens such as bacteria and mould, DNA, RNA and the like caused by soaking in a liquid phase. And the pollution to other stored umbilical cord mesenchymal stem cells caused by the leakage of the used tube cover can be avoided, so that the safety of the stored umbilical cord mesenchymal stem cells and the future clinical research and application are ensured. Meanwhile, the storage space is saved, and the cost is further reduced. Meanwhile, the umbilical cord mesenchymal stem cells prepared by the method are applied to preparation of medicines and laboratories.

[0020] Also in a Russian invention with patent number RU2416638C2 which is granted dated 20 / 04 / 2011 titled “Stem / progenitor cell recovery from amniotic membrane of umbilical cord” relates to a method for extracting stem / progenitor cells from the amniotic umbilical cord membrane, the method comprising separating the amniotic membrane from other components of the umbilical cord in vitro, culturing the amniotic membrane tissue under conditions that allow cell proliferation, and extracting stem / progenitor cells from takna cultures. In particular, the invention relates to the extraction and cultivation of stem cells having embryonic properties, such as epithelial and I or mesenchymal stem I progenitor cells, under conditions allowing the cells to undergo mitotic expansion. In addition, the invention is directed to a method for differentiating extracted stem / progenitor cells into epithelial and I or mesenchymal cells and to therapeutic applications of such stem I progenitor cells.

[0021] And another Russian invention with patent number RU2653449C2 which is granted dated 08 / 05 / 2018 titled “Enriched nutrient medium for growing cells derived from human umbilical cord tissue” provides a method of culturing isolated cells derived from human umbilical cord tissue in a culture medium containing amino acids, vitamins, salts, nucleosides, insulin, transferrin, ethanolamine and sodium selenite, where the culture medium is enriched with serum. Method includes the addition of a serum- free nutrient solution containing amino acids, vitamins, salts, nucleotides, insulin, transferrin, ethanolamine and sodium selenite, growing cells for a sufficient period of time to allow the cells to reach the desired final population density, wherein cells derived from human umbilical cord tissue are isolated from human umbilical cord tissue substantially free of blood, capable of self-renewing and replicating in culture, having differentiation potential, expressing CD13, CD90 and HLA-ABC and not expressing CD34, CD117, and HLA-DR.

[0022] Also an US invention with patent number US20180119103A1 which is granted dated 20 / 07 / 2021 titled” Isolation and cultivation of stem / progenitor cells from the amniotic membrane of umbilical cord and uses of cells differentiated therefrom” relates to a method of promoting skin rejuvenation in a subject. The method comprises administering to the subject an effective amount of a cellular extract of epithelial or mesenchymal stem / progenitor cells isolated from the amniotic membrane of the umbilical cord, wherein the cellular extract contains growth factors and peptides and is in the form of a supernatant into which the epithelial or mesenchymal stem / progenitor cells secrete the growth factors and peptides.

[0023] DESCRIPTION OF THE INVENTION

[0024] The present invention and also the method of producing the product which is a kind of lyophilized powder enriched with growth factors using conditional medium produced with the help of growth medium supplement based on the umbilical cord mesenchymal stem cells to produce the growth stimulants and repairing tissue in the body. The production method of the present inventive product includes the separation of Wharton's jelly from the human umbilical cord in the first step. At this stage, the umbilical cord of the fetus born by cesarean section is prepared under sterile conditions and in physiological serum from the hospital. The umbilical cord is washed in 70% alcohol and then the umbilical cord is cut into small pieces of 2 cm under the hood and washed with PBS and then washed in HBSS. The pieces are cut in half and their Wharton jelly is taken out and transferred to the culture medium. In the next step, the culture medium used in this invention, which is the culture medium FBS, DMEM, Pen-Strip (0.25%) and the culture medium supplement, is prepared. In order to produce the mentioned culture medium, laminar hood, incubator with the ability to adjust the CO2 concentration, invert optical microscope, Falcon centrifuge, autoclave, T25 and T75 cell culture flasks, 15 and 50 ml Falcons and microtubes, sampler and head sampler with three different sizes 10, 100 and 1000 microliters are used.

[0025] To make the said growth medium, the DMEM growth medium and fetal bovine serum (FBS) with culture medium supplement and streptomycin and Penicillin antibiotics and also Phosphate-buffered saline (PBS) and Trypsin enzyme are used.

[0026] In order to make basic cell growth medium and to provide necessary food and vital elements of the cell to growth and propagation in the laboratory medium, a growth medium is being used. Antibiotics are used to prevent contamination of the growth medium by bacteria and fungus. The DMEM growth medium should be treated by Penicillin and streptomycin antibiotics before use. The Gibco Company antibiotic is prepared with concentration of 10,000 Units / mL and is diluted pro rata 1 from 100 (500 pL in 50 mL medium) for use. The fetal bovine serum is used for providing growth factors, hormones and necessary mitogens of the cells. Without existence of these growth factors, the cells are not able to grow and propagate. Different mammalian cells in the laboratory have unique nutritional needs that must be implemented as much as possible to provide them with an optimal growth medium, a defined medium containing specific amounts of amino acids, vitamins, salts, rare elements, nucleotides and antibiotics is required in most of the laboratory culture systems, fetal bovine serum is an essential component in the growth and cultivation of cells. The fetal bovine serum is selected for its high level of growth factors, low cross-reactivity with other animal cells and high protein content. Fetal bovine serum is also used to coat the surface of the culture dish and to promote cell adhesion.

[0027] To make the culture medium supplement in the preset invention, a combination is used in the early stages of mesenchymal cell culture from the umbilical cord. The contents of the culture medium supplement includes Hepatocyte growth factor in the amount of 900-1000 ng / cc and epithelial growth factor in the amount of 15-20 ng / cc and Platelet- drived growth factor AA in the amount 2000-2500 ng / cc and Stem cofactor in the amount of 250-300 ng / cc and Insulin-like growth factor 1 in the amount of 30-50 ng / cc and Fibroblast growth factor in the amount of 30-40 ng / cc according to table 1 are used.

[0028] Using the combination of culture medium supplement with FBS makes the mesenchymal cells grow and multiply and have higher biological power compared to the culture medium without supplements. The factors in this composition are mainly mitogen and cause an extreme increase in mitotic divisions in mesenchymal cells. In the culture medium containing the supplement, the cells are small, narrow and with little cytoplasm, and most of the cells are spindle-shaped and long as shown in figure 6, while in the culture medium without the supplement, wide and spherical cells are also seen as shown in figure 5. The cells grown in the culture medium containing the supplement have a very crowded and dense appearance compared to the other group. The narrowness and spindle state of the cells is caused by the high rate of propagation and as a result of the crowding of the cell population and pushing the cells together due to the lack of sufficient space, while in the culture medium with no supplement due to the lower rate of propagation and as a result more gaps between the cells in order to reach each other to multiply and grow as well as possible and to create intercellular communication, the cells became wide and ramified and this may cause progeria of the cells. In the present invention, 20% of the FBS culture medium and 20-25% of the supplement composition of the culture medium are used to prepare the complete culture medium. The formula for preparing the complete final culture medium for the primary culture of human umbilical cord mesenchymal cells as shown in table 2 includes 20% fetal bovine serum (FBS) plus 20-25% culture medium supplement, 1% penicillin- streptomycin antibiotic and 44-49% DMEM culture medium. After preparation, the final solution is passed through a nano silver filter to filter out any fungal, bacterial and viral contamination and use the final product as a sterile solution. To make the DMEM culture medium, first 450 mL sterile distilled water pulled into a sterile beaker, placed a magnet inside it, and turned on the stirrer at low speed so that the magnet started rotating. Then, 6.7 gr DMEM powder added smoothly to the distilled water and after complete dissolving of the medium, 1 gr sodium bicarbonate powder (1.85 gr) added. After the complete dissolution of the powder, while the magnet is rotating, the pH of the medium was adjusted to 7.2 to 7.4 with the help of HC1, and in the last step, the volume of the environment to 500 ml increased.

[0029] Immediately the medium should be transferred under the Laminar flow hood where all the equipment is sterilized by UV light for 20 minutes, and sterilize it next to the flame of the alcohol lamp with a needle and poured it into the container with a screw door and this medium can be kept at a temperature of +4 degrees Celsius for a month as the main stock of the basic culture medium. The PBS solution is used to wash the cells and eliminate the death cells. In order to prepare PBS solution, prepared tablets were used. For this purpose, a PBS tablet was dissolved in 200 ml of sterile distilled water and autoclaved according to the company's protocol. Trypsin enzyme is also used in the desired solution. This enzyme is used to separate the cells which are stick to the bottom of the culture vessel. Cells stick to the bottom of the vessel with glycoprotein bonds, trypsin enzyme breaks the amino acid bond of proteins after the trypsin amino acid, causing the cells to detach. Depending on the type of cell category, the time required for cell separation is different. In order to prepare 50 ml of trypsin enzyme, the distilled water poured into an Erlenmeyer flask and put a magnet inside it. Then we turned on the stirrer so that the magnet rotates. 2.5 mg of trypsin powder was poured into Erlenmeyer flask and waited for it to dissolve. Then 100 microliters of EDTA solution added and sterilized it with a filter. The stock kept at -20°C. In the following, condition medium is prepared from Wharton's jelly stem cells. Factors secreted from stem cells can be identified in the environment where stem cells are cultured, which is called conditional medium or conditioned medium. Since the conditioned medium does not contain cells, it prevents the occurrence of immune reactions and there is no problem of transplant rejection. Also, the conditioned medium has an antioxidant and antitumor effect and contains different growth factors and cytokines that are secreted by various stem cells, including: VEGF, PDGF, EGF, HGF, NGF, BDNF, IGF-I, IGF-II, KGF, bFGF, IL-6 and other growth factors secreted by stem cells are present in the conditioned medium, the number of cells, type of cells, culturing condition, type of culturing medium and number of passages affects the quality of conditioned medium. The conditioned medium has lots of advantages such as being easily produced, frozen, packaged and transferred that for these features the conditioned medium is used to make medicines and it also does not require special protection, such as what should be observed in the case of stem cells. On the other hand, unlike stem cells, which have poor grafting and the possibility of cancer spreading in them is high, but the conditional environment does not have these problems, and it also has the advantage that it can be used to treat patients (clinically) quickly without separating the stem cells from the patient. In order to collect the conditional medium of the Wharton’s jelly stem cells, when the cultured cells in the second passage occupy about 90% of the surface of the culture medium and there is no distance between the cells, the culture casing of culture medium is discarded and washed twice with PBS, then the culture medium without FBS is added and the flasks are transferred to the incubator. After 48 hours and casing medium of the cells collected and poured in to the Falcon tube and is centrifuged at 2000 rpm for 5 minutes. Then in order to prevent the existence of any dead cells and piece of cells, the conditioned medium is filtered first with 0.22 micrometer syringe filter and then with nanosilver filter for disinfection, the medium is aliquoted in a Falcon tube and transferred to a -80 freezer for storage. The most important feature of the conditioned medium of the stem cells is the short lifetime and low durability in the normal temperature and it should be frozen in the temperature about -80 for long term storage. This makes the use of conditioned medium faced with serious limitations. In order to increase the durability and the ability to store at room temperature, as well as to use it in pharmaceutical products, lyophilized powder is obtained from the conditioned medium of stem cells using the freeze-drying method. In this method the durability of the product increases up to 2 years at room temperature and up to 3 years at refrigerator temperature (4 degrees). Also, it can be easily transported due to its very small volume and sterile packaging. So that one liter of conditioned medium, which should be kept in a freezer at -80 degree, turns into 7 grams of lyophilized powder, which can be stored and transported at room temperature for a long time.

[0030] To prepare the lyophilized powder, the conditioned medium liquid samples that are inside the flask are transferred to the Christ-alpha model freeze-dryer (made in Germany). Lyophilization is done at a temperature of -50 degrees centigrade. The pressure level of the device starts from 0.7 millibar at the begining and reaches 0.03 millibar at the end of the process, and the average pressure value is calculated 0.4 millibar. The duration of placement of the sample in the device is 24 to 48 hours, depending on the sample volume. In the final phase, the enrichment of the lyophilized powder of conditioned medium is done by using additives. These additives are in liquid form and are used as a solvent for the lyophilized powder before consumption. According to the final use of the product, the type of additives will be different.

[0031] DESCRIPTION OF SOME PRACTICAL METHODS

[0032] In one practical method in order to prepare a formula for mesotherapy, the following example was done as the amount and percentage of mixing materials and preparation of mesotherapy medicine using conditioned medium base. The amount of 2-4 ng / ml Vascular endothelial growth factor (VEGF-A), the amount of 5-10 ng / ml Fibroblast growth factor (FGF-1), the amount of 1-5 ng / ml Epidermal growth factor (EGF), the amount of 10-15 ng / ml Insulin-like growth factor- 1(IGF- I), the amount of 60-80 ng / ml L-Cystine»2HCl, the amount of 500-600 ng / ml L- Glutamine, the amount of 100-150 ng / ml L-Isoleucine, the amount of 150-180 ng / ml L- Leucine, the amount of 100-150 ng / ml L-Lysine»HCl, the amount of 4-5 ng / ml Folic Acid, the amount of 4-5 ng / ml Nicotinamide, the amount of 4-5 ng / ml D-Pantothenic Acid, the amount of 4-5 ng / ml Riboflavin and the amount of 4-5 ng / ml Thiamine’HCl are used in this example according to table 3.

[0033] In one practical method in order to prepare a formula for skin rejuvenation, the following example was done as the amount and percentage of mixing materials and preparation of skin rejuvenation medicine using conditioned medium base. The amount of 2-4 ng / ml Vascular endothelial growth factor (VEGF-A), the amount of 10-20 ng / ml Fibroblast growth factor (FGF-1), the amount of 1-5 ng / ml Epidermal growth factor (EGF), the amount of 5-10 ng / ml Insulin-like growth factor- 1(IGF- 1), the amount of 60-80 ng / ml L-Cystine»2HCl, the amount of 500-600 ng / ml L- Glutamine, the amount of 100-150 ng / ml L-Isoleucine, the amount of 150-180 ng / ml L- Leucine, the amount of 100-150 ng / ml L-Lysine»HCl, the amount of 3-4 ng / ml Folic Acid, the amount of 4-5 ng / ml Nicotinamide, the amount of 5-6 ng / ml D-Pantothenic Acid, the amount of 4-5 ng / ml Riboflavin and the amount of 4-5 ng / ml Thiamine*HCl are used in this example according to table 4.

[0034] In one practical method in order to prepare a formula for repair of skin wounds, the following example was done as the amount and percentage of mixing materials and preparation of repair of skin wounds medicine using conditioned medium base. The amount of 2-4 ng / ml Vascular endothelial growth factor (VEGF-A), the amount of 10-20 ng / ml Fibroblast growth factor (FGF-1), the amount of 2-3 ng / ml Epidermal growth factor (EGF), the amount of 5-10 ng / ml Insulin-like growth factor- 1 (IGF- 1), the amount of 60-80 ng / ml L-Cystine*2HCl, the amount of 500- 600 ng / ml L-Glutamine, the amount of 100-150 ng / ml L-Isoleucine, the amount of 150-180 ng / ml L-Leucine, the amount of 100-150 ng / ml L-Lysine’HCl, the amount of 4-5 ng / ml Folic Acid, the amount of 4-5 ng / ml Nicotinamide, the amount of 5-6 ng / ml D-Pantothenic Acid, the amount of 4-5 ng / ml Riboflavin and the amount of 4-5 ng / ml Thiami ne*HCl are used in this example according to table 5.

[0035] Extraction of conditioned medium from umbilical cord mesenchymal cells is being done in different countries, but with different techniques and efficiency, however the goals of all these techniques include the following:

[0036] 1. Increasing the production efficiency 2. Increase of undifferentiated small spindle-shaped cells known as Rapid Cycle to secrete more and more CM

[0037] 3. Reduction of large spherical differentiated cells known as Slow Cycle

[0038] 4. Increasing the richness of CM content

[0039] 5. Reduction of production time for commercial production

[0040] 6. Complete removal of dead cells and removal of fungal, bacterial and viral contamination using nano silver filter

[0041] 7. Increasing durability and ability to store at room temperature

[0042] In table 6, the reasons for the preference of the technique used in this research over other common techniques in the comparison are explained.

[0043] BRIEF DESCRIPTION OF DRAWINGS

[0044] Figure 1 is a flowchart showing the amount of components comprises the supplement of culture medium.

[0045] Figure 2 is a flowchart showing the steps and components of the culture medium.

[0046] Figure 3 is the flowchart of the steps of separating the human umbilical cord from Wharton's jelly.

[0047] Figure 4 is the flow chart of preparation steps of lyophilized powder enriched with growth factors.

[0048] Figure 5 shows spherical cells in culture medium without supplements. Figure 6 shows the spindle-shaped cells in the culture medium containing the supplement.

[0049] Table 1 shows growth factors and their average amount in culture medium.

[0050] Table 2 shows the formula for preparing the complete final culture medium for the primary culture of human umbilical cord mesenchymal cells.

[0051] Table 3 shows the formula for preparing hair mesotherapy solution.

[0052] Table 4 shows the formula for preparing the skin rejuvenation solution.

[0053] Table 5 shows the formula for the preparation of skin wound healing solution.

[0054] Table 6 shows the reasons for the preference of the used technique over other common techniques.

Claims

What is claimed is1. The invention of product and method of producing lyophilized powder enriched with growth factors using conditioned medium produced with the help of growth medium supplement based on umbilical cord mesenchymal stem cells for the production of growth stimulants and tissue repair in the human body including at least one culture medium based on DMEM and FBS with growth supplement and at least one antibiotic to sterilize the growth medium and at least one enzyme to separate the cells from the culture container and at least one filtration step with a membrane filter and at least two culture passages and at least production of a conditioned medium and at least one step of filtering the culture medium and at least one step of lyophilization of the conditioned medium and at least one group of umbilical cord stem cells.

2. The product and method of claim 1 which uses stem cells in Wharton's umbilical cord gel as primary cells to produce lyophilized powder.

3. The product and method of claim 1 in which in order to extract Wharton's gel, the umbilical cord is first washed with 70% alcohol and then washed again with PBS and HBSS.

4. The product and method of claim 1 in which the culture medium is prepared from FBS, DMEM, Pen-strip 0.25%, along with PBS buffer and antibiotics, and the culture medium supplement is added to create the final culture medium.

5. The product and method of claim 1 in which the culture medium supplement includes fibroblast growth factor with a concentration of 30-40 ng / cc along with insulin-like growth factor with a concentration of 30-50 ng / cc along with hepatocyte growth factor with a concentration of 900-1000 ng / cc and epithelial growth factor with a concentration of 15-20 ng / cc along with platelet-derivedgrowth factor with a concentration of 2000-2500 ng / cc along with stem cell factor with a concentration of 250-300 ng / cc.

6. The product and method of claim 1 in which the use of culture medium supplements causes mesenchymal cells to grow and multiply and have higher biological power compared to the culture medium without supplements.

7. The product and method of claim 1 in which the growth supplement causes a sharp increase in mitotic divisions in mesenchymal cells.

8. The product and method of claim 1 that due to the use of growth supplements, spindle-shaped and elongated cells are produced and multiplied far more than spherical cells.

9. The product and method of claim 1 which, due to the higher growth rate of mesenchymal cells, they don’t have the opportunity to fill the empty spaces with the help of widening, and the possibility of progeria in the multiplied cells is reduced.

10. The product and method of claim 1 in which the final culture medium is cleaned from any residual cells or disturbing microorganisms by membrane filtration in order to reduce contamination and increase efficiency.

11. The product and method of claim 1 which uses trypsin enzyme in the culture medium to prevent cells from sticking to the walls of containers.

12. The product and method of claim 1 which by multiplying stem cells, creates a conditioned medium in the culture medium, and the factors secreted by the stem cells are concentrated in the culture medium.

13. The product and method of claim 1 that the conditioned medium is sterilized and filtered in the second passage with the help of a membrane filter equipped with Nano silver.

14. The product and method of claim 1 that the conditional medium containing the growth factors secreted by the multiplied stem cells turned into powder in the lyophilizer and at a temperature of -80 degrees Centigrade.

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