LU502428B1 - Polysaccharide Biomedical colloid Liquid enriched with the factor complex of human mesenchymal stem cells from adipose tissue, and its production and use - Google Patents

Abstract

The present invention relates to a polysaccharide biomedical colloid liquid which is enriched with the factor complex of human mesenchymal stem cells from adipose tissue, and its production and use, comprising the following steps: S1, purification of adipocytes; S2, culturing mesenchymal stem cells from adipose tissue (hASCs); S3, collecting supernatant before passage; S4, preparing a complex biomedical colloid liquid; Stem Cell Complex Factor Concentrate is formulated with sodium carboxymethyl cellulose and pectin to create a moist environment on the wound surface that promotes granulation and re-epithelialization of the wound surface and forms a protective film on the wound surface that prevents bacterial infection of the wound surface and the process of adsorption , Proliferation and replication of bacteria on the wound surface inhibits. The biomedical colloid is also fluid and malleable, making it suitable for application to any part of the patient's body.

Description

Description Polysaccharide biomedical colloid liquid enriched with 4502428 factor complex of human adipose tissue mesenchymal stem cells and its production and use Technical Field The present invention belongs to the technical field of new uses of stem cells and relates to a polysaccharide biomedical colloid liquid enriched with the factor complex enriched in human mesenchymal stem cells from adipose tissue, and their production and use.

Background Technology Human adipose-derived mesenchymal stem cells (hASC) are a class of pluripotent stem cells with the potential for self-renewal and multidirectional differentiation. It has been found that human adipose-derived mesenchymal stem cells can be used to treat a variety of diseases, with their therapeutic role often being attributed to their cytokine secretion and immunomodulatory effects rather than their ability to differentiate into target cells.

The supernatant of the conditioned medium of human adipose-derived mesenchymal stem cells contains a variety of growth factors, cytokines, chemokines, angiogenic factors, and exosomes that significantly promote cell proliferation, migration, and differentiation. In addition, it is easier to industrially manufacture, freeze, store, package and transport the cells than direct application of stem cells. In addition, the supernatant is relatively safe to use as it does not contain cells and is therefore not rejected by the immune system.

Wound healing is a complex biological process that includes acute wound healing and chronic wound healing. Studies have shown that growth factors promote granulation tissue formation in acute wounds while promoting re-epithelialization and 1

Accelerate Description. In the treatment of chronic refractory wounds, the use of recombinant human platelet growth factor and fibroblast growth factor significantly promotes granulation and re-epithelialization of wounds and has been shown to be effective in the treatment of clinically refractory diabetic foot ulcers, decubitus ulcers and other wounds. Although high doses of a single factor can be used to some extent to treat refractory wounds, its clinical application is limited by its single regulatory mechanism, complicated purification process, and high cost.

The traditional method of treating wounds with growth factors is to spray the solution onto the wound. This has the advantage that the growth factors act quickly and evenly on the repair tissue, but the easy loss and evaporation of the solution limits the potency and stability of the growth factors to some extent.

Therefore, the development of a stem cell factor complex enriched with stem cell factors derived from the supernatant of the culture medium for human mesenchymal stem cells and supported by a better adjuvant to create a moist environment for the trauma surface that allows the growth factors to be consistently and evenly distributed to act on the trauma tissue shows great promise for clinical application. SUMMARY OF THE INVENTION In view of the above, the present invention provides a polysaccharide biomedical colloid liquid enriched in human adipose tissue mesenchymal stem cell factor complex, and its manufacture and use to solve the above problems of the prior art in acute and chronic Wounds to 16s associated with many adverse reactions, cumbersome handling and limited application.

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Description

In order to achieve the above objectives, the present invention offers the following technical solutions:

A process for the preparation of a polysaccharide biomedical colloid liquid enriched in the factor complex of human adipose tissue mesenchymal stem cells, comprising the following steps:

S1. Purifying Adipocytes

Collecting human fat samples, washing with saline, adding an equal volume of collagenase to digest, filtering and centrifuging several times after digestion, and removing the supernatant to obtain purified adipocytes;

S2. Culturing Mesenchymal Stem Cells from Adipose Tissue (hASCs)

The purified adipocytes were resuspended in serum-free phenol red-free High Coomassie medium containing 5% platelet lysate and then transferred to cell culture flasks for incubation at an inoculation density of 60,000 cells/ml; Culture mesenchymal stem cells from adipose tissue under hypoxic conditions to 90 ± 5% to promote cytokine secretion and collect the culture supernatant between generations P3 and P5;

S3. Collect supernatant before passage

The supernatant before the passage was centrifuged at high speed by a centrifuge and concentrated to prepare a cell culture broth, which was filtered through a filter membrane with a diameter of 0.22 µm and debacterized to finally obtain the stem cell complex factor concentrate;

S4. Manufacture of a complex biomedical colloidal fluid

Mix sodium carboxymethyl cellulose and pectin in a molar ratio of 80-90:1 to obtain a hydrocolloid gel of 1 ml, then dilute the hydrocolloid gel to 5-10 times its volume with normal saline, and then add a stem cell complex factor concentrate ( concentration of 10ug-20ug/L),

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Description to obtain a complex biomedical colloid liquid. LUS02428 The collagenase in step S1 is a mixture of type I collagenase and type II collagenase, the digestion time is 30-60 minutes, the collagenase concentration is 0.1% to 0.5%, the digested adipocytes are filtered through a membrane with a diameter of 70 µm and then are centrifuged at 2000rpm/min for 5 minutes to remove the supernatant, and the cell precipitate was then dispersed in PBS under the same conditions, and the purified adipocytes were recovered by filtration and centrifugation again.

The serum-free phenol red-free High Coomassie medium in step S2 is a mesenchymal stem cell medium supplemented with 10 mg to 28 mg coenzyme A, 5 mg to 15 mg co-carboxylase, 4 mg to 12 mg flavin adenine dinucleotide and 0.5 mg to 2.5 mg uridyl phosphate per 1L supplemented; The hypoxic culture conditions are: the oxygen concentration of the cells is 21% in 24 hours, from 24 to 26 hours the oxygen concentration decreases steadily from 21% to 18%, in the 27 hour the oxygen concentration is 18%, from the 28 to 36 hour the Oxygen concentration decreases smoothly from 18% to 8%, in the 37 hour the oxygen concentration is 8%, from 38 to 44 hours the oxygen concentration decreases smoothly from 8% to 2%, and then continues to incubate at an oxygen concentration of 2%, where the CO2 concentration is 5% and the rest is nitrogen.

The secretions obtained in culture during the recovery of the extracted mesenchymal stem cells from adipose tissue in step S2 contain at least vascular endothelial growth factor (VEGF), keratinocyte growth factor (KGF), hepatocyte growth factor (HGH), transforming = growth factor β (TGF-B), insulin-like growth factor (IGF) and platelet-derived growth factor (PDGF).

Human mesenchymal stem cells derived from adipose tissue secrete vascular endothelial growth factor (VEGF), the keratinocyte-4

Description Growth factor (KGF), hepatocyte growth factor (HGH), PB transforming growth factor (TGF-B), insulin-like growth factor (IGF) and platelet-derived growth factor (PDGF). The main biological activities of these cytokines are anti-inflammatory, anti-apoptotic and immunomodulatory. They strongly promote mitosis, are involved in stem cell-induced differentiation, tissue cell growth, regeneration and reconstruction, improve tissue function at the site of trauma, and promote wound healing.

Vascular endothelial growth factor (VEGF) was between

728.56+124.23pg/ml, the keratinocyte growth factor (KGF) was between

623.46+136.78pg/ml, the hepatocyte growth factor (HGH) was between

2501.11+18.36pg/ml, transforming growth factor B (TGF-B) was between 4223.23+22036pg/ml, insulin-like growth factor (IGF) was between 1653.56+56.23pg/ml and platelet-derived growth factor (PDGF) was between 102.56+ 23.53pg/ml.

The sodium carboxymethyl cellulose in step S4 is a mixture of low-viscosity sodium carboxymethyl cellulose, medium-viscosity sodium carboxymethyl cellulose and high-viscosity sodium carboxymethyl cellulose in a molar ratio of 1:1:1.

Polysaccharide Biomedical Colloid Human Adipose-derived Mesenchymal Stem Cell Factor Complex-enriched Liquid prepared by the Process for the Production of Human Adipose-derived Mesenchymal Stem Cell Factor Complex-Human Adipose-derived Mesenchymal Stem Cell Factor Complex and Polysaccharide Biomedical Colloid liquid combined.

Use of a polysaccharide biomedical colloid liquid made with a factor complex derived from human mesenchymal stem cells

Description Adipose tissue enriched in the treatment of acute and chronic*°°428 wounds.

The beneficial effects of the present invention are:

1. The invention discloses a polysaccharide biomedical colloid liquid enriched with human adipose tissue mesenchymal stem cell factor complex, stem cell complex factor concentrate as the core content of the treatment of trauma, can effectively and quickly repair acute and chronic wounds, and the repair process has low side effects and quick results, filling the gap in the market to directly repair acute and chronic wounds of topical drugs. Stem Cell Factor Concentrate is formulated with sodium carboxymethyl cellulose and pectin to create a moist environment on the traumatic surface. It is used for cell repair to form a protective film on the trauma surface, which blocks bacterial infection of the trauma surface and inhibits the process of bacterial adsorption and proliferation on the trauma surface. At the same time, this biomedical colloid is fluid and malleable, making it suitable for application to any part of the patient's body.

2. Polysaccharide Biomedical Colloid Liquid enriched with human adipose tissue mesenchymal stem cell factor complex disclosed in the present invention promotes the repair of scalded wounds with obvious effect, the epidermis of the wounds is intact and thick after 7 days of application, the subcutaneous collagen is neatly arranged; Hair follicles and blood vessels were regenerated regularly and more frequently.

3. Polysaccharide Biomedical Colloid Liquid enriched with human adipose tissue mesenchymal stem cell factor complex disclosed in the present invention is the concentrated adipose tissue mesenchymal stem cell culture supernatant preparation of human origin and has no 6

Description germ line problems; The finished product contains no live/>92428 cell components, no ethical or immunological safety issues, no need for ready-to-use preparation, and ease of use.

Additional advantages, objects, and features of the present invention will be set forth in part in the description that follows, and in part will be apparent to those skilled in the art based on examination of the following study, or may be learned from practice of the present invention. The objectives and other advantages of the present invention may be attained and attained by the use of the following description.

Description of the attached drawings In order to clarify the object, the technical solutions and the advantages of the present invention, the invention is preferably described in more detail below in connection with the attached drawings, in which: Figure 1 shows a flow chart of a process for the production of a polysaccharide biomedical colloid Fluid enriched with the factor complex of human mesenchymal stem cells derived from adipose tissue. DETAILED DESCRIPTION Example 1 A method of preparing a polysaccharide biomedical colloid liquid enriched in human adipose tissue mesenchymal stem cell factor complex as shown in Figure 1, comprising the steps of: S1. purifying adipocytes; collecting human fat samples, washing with saline, adding an equal volume of collagenase for digestion, filtering and centrifuging several times after digestion and removing the supernatant to obtain purified adipocytes; The collagenase is a mixture of type I collagenase and 7

Description is type II collagenase, the digestion time is 30-60 minutes, the collagenase/*02428 concentration is 0.1% to 0.5%, the digested adipocytes are filtered through a membrane with a diameter of 70um, and then at 2000rpm/min 5 - Centrifuged for minutes to remove the supernatant, and the cell precipitate was then dispersed in PBS under the same conditions, and the purified adipocytes were recovered by filtration and centrifugation again.

S2. Cultivation of Adipose Tissue Mesenchymal Stem Cells (hASCs) The purified adipocytes were resuspended in serum-free phenol red-free High Coomassie medium containing 5% platelet lysate and then transferred to cell culture flasks for incubation at an inoculation density of 60,000 cells/ml; The serum-free phenol red-free high-Coomassie medium is a mesenchymal stem cell medium supplemented with 10 mg to 28 mg coenzyme A, 5 mg to 15 mg co-carboxylase, 4 mg to 12 mg flavin adenine dinucleotide and 0.5 mg to 2.5 mg uridyl phosphate per 1L supplemented; Cultured under hypoxic conditions to promote the secretion of cytokines, mesenchymal stem cells from adipose tissue were cultured at 90±5%, and the culture supernatants of the P3 generation were collected; The hypoxic culture conditions are: the oxygen concentration of the cells is 21% in 24 hours, from 24 to 26 hours the oxygen concentration decreases smoothly from 21% to 18%, in the 27 hour the oxygen concentration is 18%, from 28 to 36 hours it increases Oxygen concentration decreases smoothly from 18% to 8%, in the 37 hour the oxygen concentration is 8%, from 38 to 44 hours the oxygen concentration decreases smoothly from 8% to 2%, and then continues to incubate at an oxygen concentration of 2%, where the CO2 concentration is 5% and the remainder is nitrogen.

The secretions include at least vascular endothelial growth factor (VEGF), keratinocyte growth factor (KGF), hepatocyte growth factor (HGH), transforming growth factor B (TGF-B), insulin-like growth factor (IGF), and platelet-derived 8th

Description derived growth factor (PDGF). LUS02428 Human adipose-derived mesenchymal stem cells secrete vascular endothelial growth factor (VEGF), keratinocyte growth factor (KGF), hepatocyte growth factor (HGH), transforming growth factor PB (TGF-B), insulin-like growth factor (IGF) and platelet-derived growth factor (PDGF). The main biological activities of these cytokines are anti-inflammatory, anti-apoptotic and immunomodulatory. They strongly promote mitosis, are involved in stem cell-induced differentiation, tissue cell growth, regeneration and reconstruction, improve tissue function at the site of trauma, and promote wound healing. Vascular endothelial growth factor (VEGF) was between

728.56+124.23pg/ml, the keratinocyte growth factor (KGF) was between

623.46+136.78pg/ml, the hepatocyte growth factor (HGH) was between

2501.11+18.36pg/ml, transforming growth factor B (TGF-B) was between 4223.23+=220 36pg/ml, insulin-like growth factor (IGF) between 1653.56+56.23pg/ml and platelet-derived growth factor (PDGF) between 102.56 +23.53pg/ml.

S3. Collection of the supernatant before the passage The supernatant before the passage was centrifuged at high speed by a centrifuge and concentrated to prepare a cell culture broth, which was filtered through a filter membrane with a diameter of 0.22 µm and debacterized to finally obtain the stem cell complex factor concentrate; S4. Preparing complex biomedical colloid liquid Mix sodium carboxymethyl cellulose and pectin in a molar ratio of 80-90:1 to obtain a hydrocolloid gel of 1ml, then dilute the hydrocolloid gel to 5-10 times its volume with physiological saline, and then add a 9

Description Stem cell complex factor concentrate (concentration of 10ug-20ug/L) hinztY>92428 to obtain complex biomedical colloid liquid.

Example 2 The difference between Example 2 and Example 1 is that the sodium carboxymethyl cellulose in step S4 is a mixture of low viscosity sodium carboxymethyl cellulose, medium viscosity sodium carboxymethyl cellulose and high viscosity sodium carboxymethyl cellulose in a molar ratio of 1:1:1.

Example 3 The difference between example 3 and example 2 is that in step S2 the supernatant collected is the supernatant of the P4 generation.

Example 4 The difference between example 4 and example 2 is that in step S2 the supernatant collected is the supernatant of the P5 generation.

Example 5 The difference between example 5 and example 2 is that in step S4 the concentration of the stem cell complex factor concentrate is 15 µg/L.

Example 6 The difference between Example 6 and Example 2 is that in step S4, the concentration of the stem cell complex factor concentrate is 20 µg/L.

Comparative Examples The biomedical polysaccharide colloid solution contains in weight percent: low viscosity sodium carboxymethyl cellulose 0.3%, medium viscosity sodium carboxymethyl cellulose 1.2%, high viscosity sodium carboxymethyl cellulose 0.4%, sodium chloride 0.9%, the balance being water for injection. Dissolve sodium chloride in water for injections, then low, medium and high viscosity sodium carboxymethyl cellulose

Description into the water, turn on the thermostat and stir mechanically at a temperature of vdrr°02428 65°C until the low, medium and high viscosity sodium carboxymethyl cellulose is completely dissolved to obtain a biomedical colloidal polysaccharide solution.

The finished products produced in Examples 1 to 6 were tested on rats in the following manner:

1. Rat Acute Trauma Model: At least 18 adult SD rats were collected and divided into 6 treatment groups, 3 rats in each group. 12 hours before modeling, remove the hair on the rats' backs, fast and drink plenty of water. Prior to modeling, rats were anesthetized by intraperitoneal injection of chloral hydrate solution and the dorsal hair removal area was routinely disinfected. A circular area (distance between circular marks was >1 cm) 2 cm in diameter was marked on each side of the spinal column of each rat, and the entire skin was excised along the circular marks with a sterile scalpel, making the wound deep into the deep fascia should be enough.

2. Treatment method: The wounds on the left and right side of the rat back were set up as treatment and control groups, respectively. The finished products 1 to 6 were applied to the skin wounds of the respective groups of rats, and the wounds were cleaned with sterile saline and replaced with new dressings after disinfecting the wounds with amyl iodine every 2 days. During treatment, the rats were observed for tissue repair and other responses such as diet and exercise.

3. Evaluation of Treatment Effects After treatment, the rats were excised and all skin and some random subcutaneous tissue samples were taken from the injured area and sent to pathological section, and each sample was stained with hematoxylin-eosin; 11

Description The test results are as follows: LUS02428 The rats of Examples 1-6 had an intact and thick epidermis with an ordered collagen arrangement; Hair follicles were present, blood vessels grew regularly and were more numerous; subcutaneous collagen was produced and properly assembled, and the results were better than the inverse ratio.

Examples 1-6 and the scale of restoration are presented in Table 1, where + represents the degree of restoration, the more + the better the restoration, and - for no restoration.

Table 1 [ee [rn [ee Infiltration Example | | he | fo | Example 2 | eer | SH NE Example 3 | ee (OT fo | Example 4 | er | oe fo | TE | Examples SE OT EE NE EET | OT (OT UOTE Comparative + + + + + [rés LIL Note: The number of "+" indicates the degree of tissue repair after the treatment on.

The finished products produced in Examples 1 to 6 were tested on rats in the following manner:

1. Implementation of chronic trauma model in rats with difficult healing At least 21 adult SD rats were taken and divided into 7 groups, 1 control group and 6 chronic trauma groups, 3 rats in each group anesthetized by intraperitoneal injection of chloral hydrate solution prior to modeling became. The dorsum was debrided and the 12th

The impression was marked with a 3.0 cm x 3.0 cm card that was fixed with sutures around the 502428 debrided area. Prepare a sterile disposable incision set and other utensils, and after the anesthesia has taken effect, disinfect the skin preparation area of the rat's back with iodophor three times, cut away the subcutaneous tissue along the marked line up to the myofascia, stop the bleeding and make up an open full-thickness skin defect in the rat.

2. Treatment method: Immediately after the shaping, hydrocortisone (80 mg/kg) was injected intramuscularly for 7 days. 2 ml of the finished product prepared in Examples 1 to 6 were applied to the wound. Penicillin potassium salt (4,000 units) was injected intramuscularly for 4 days from 1 day before the experiment to prevent infection. Wounds were changed every other day, routinely irrigated and bandaged. During treatment, the rats were observed for tissue repair and other responses such as diet and exercise.

3. Determination of Treatment Effects The size of the wound is recorded using transparencies and photographic methods, and some animal wounds are processed and calibrated using a computerized image analysis system to calculate the speed and quality of wound healing. In addition, the rats were executed at the end of the treatment, and all the skin and part of the attached subcutaneous tissue were taken out from the injured area and sent to the pathological section, and each specimen was stained with hematoxylin-eosin.

The test results are as follows: Examples 1-6 Rats with red and wet trauma, little secretion, irregular shape of the trauma margin and clear contraction.

Examples 1-6 and the scale of repair are shown in Table 1, 13

Description where + stands for the level of repair, the more +, the better the repair effect/202428 - stands for no repair.

TS TEE infiltration example | ee | | | (eer |e [ee [ee | Examples | ew | ew | TE EEE I UOTE | 8 | Example 5 | ev UOTE 2 om | | Bewielo | we | oe UT EL | | CV 8181 + + + + + Examples Finally let noted that the above embodiments only serve to illustrate the technical solutions of the present invention and not limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention modified or equivalently substituted without departing from the spirit and scope of the technical solutions, all of which should be included within the scope of the claims of the present invention.

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Claims (7)

claims 1. Process for the production of a polysaccharide biomedical colloid°°2%28 liquid which is enriched with the factor complex of human mesenchymal stem cells from adipose tissue, characterized in that it comprises the following steps: S1. Purification of adipocytes Taking human fat samples, washing with saline, adding an equal volume of collagenase for digestion, filtering and centrifuging several times after digestion, and removing the supernatant to obtain purified adipocytes; S2. Cultivation of Adipose Tissue Mesenchymal Stem Cells (hASCs) The purified adipocytes were resuspended in serum-free phenol red-free High Coomassie medium containing 5% platelet lysate and then transferred to cell culture flasks for incubation at an inoculation density of 60,000 cells/ml; Culture mesenchymal stem cells from adipose tissue under hypoxic conditions to 90 ± 5% to promote cytokine secretion and collect the culture supernatant between generations P3 and P5; S3. Collection of the pre-passage supernatant The pre-passage supernatant was centrifuged at high speed by a centrifuge and concentrated to prepare a cell culture broth, which was filtered through a filter membrane of 0.22 µm in diameter and debacterized to finally obtain the stem cell complex factor concentrate; S4. Preparation of complex biomedical colloid liquid Mix sodium carboxymethyl cellulose and pectin in a molar ratio of 80-90:1 to obtain a hydrocolloid gel of 1 ml, then dilute the hydrocolloid gel to 5-10 times its volume with physiological saline and then add add a stem cell complex factor concentrate (concentration of 10ug-20ug/L) to obtain a complex biomedical colloid liquid. 1 claims 2. Preparation method according to claim 1, characterized in that d}4/502428 collagenase in step SI is a mixture of type I collagenase and type II collagenase, the digestion time is 30-60 minutes, the collagenase concentration is 0.1% to 0.5% %, the digested adipocytes were filtered through a 70 µm diameter membrane and then centrifuged at 2000rpm/min for 5-10 minutes to remove the supernatant, and the cell precipitate was then dispersed in PBS under the same conditions, and the purified adipocytes were recovered by filtration and recentrifugation. 3. Production method according to claim 2, characterized in that the serum-free phenol red-free High Coomassie medium in step S2 is a mesenchymal stem cell medium which is supplemented with 10 mg to 28 mg coenzyme A, 5 mg to 15 mg co-carboxylase, 4 mg to 12 mg flavin adenine dinucleotide and 0.5 mg to 2.5 mg uridyl phosphate per 1L; The hypoxic culture conditions are: the oxygen concentration of the cells is 21% in 24 hours, from 24 to 26 hours the oxygen concentration decreases steadily from 21% to 18%, in the 27 hour the oxygen concentration is 18%, from the 28 to 36 hour the Oxygen concentration decreases smoothly from 18% to 8%, in the 37 hour the oxygen concentration is 8%, from 38 to 44 hours the oxygen concentration decreases smoothly from 8% to 2%, and then continues to incubate at an oxygen concentration of 2%, where the CO2 concentration is 5% and the rest is nitrogen. 4. Preparation method according to claim 3, characterized in that the secretions obtained during the extraction of the extracted mesenchymal stem cells from adipose tissue in step S2 in culture, at least the vascular endothelial growth factor (VEGF), the keratinocyte growth factor (KGF), the hepatocyte growth factor (HGH), transforming growth factor B (TGF-B), insulin-like growth factor (IGF) and platelet-derived growth factor (PDGF). 2 claims 5. Production method according to claim 1, characterized in that di&'°02428 sodium carboxymethyl cellulose in step S4 is a mixture of low-viscosity sodium carboxymethyl cellulose, medium-viscosity sodium carboxymethyl cellulose and high-viscosity sodium carboxymethyl cellulose in a molar ratio of 1:1:1 1st. 6. Polysaccharide biomedical colloid liquid enriched in human adipose tissue mesenchymal stem cell factor complex prepared by the process for preparing a polysaccharide biomedical colloid liquid enriched in human adipose tissue mesenchymal stem cell factor complex as in any one of claims 1 to 5 claimed, characterized in that the adipose mesenchymal stem cell factor complex and the biomedical polysaccharide colloid solution are fused. 7. Use of a polysaccharide biomedical colloid liquid enriched with a factor complex of human mesenchymal stem cells from adipose tissue in the treatment of acute and chronic wounds according to claim 6. 3