LV13763B - Method for preparation of autological stem cell population from dermal tissues of adult human skin - Google Patents

Abstract

Invention pertains to field of preparation, propagation and characteristic of stem cells, particularly to the preparation of multipotent autologous stem cells from dermal tissues of human skin, propagation of obtained cells and characterization of quality thereof. The offered method for preparation of multipotent stem cell population comprises cultivation of stem cells isolated from derma of adult human skin or part thereof in conditions where at least 10 % of multipotent stem cells growth and propagate adherently, but cells that belong not to family of stem cells, are separated. The cultivation of cells are carried out in medium formed from culture medium DMEM, or DMEM and F-12 in the range ratio 3:1, together with 10 % of calf serum and penicillin and streptomycin (100 u/ml and 100 microgram/ml) with or without 4 nano-gram/ml supplements of FGF-2. The offered method for characterization of cell-quality is characterized in that at least 95 % of stem cells isolated from dermal tissues of adult human skin or part thereof have surface markers: CD73, CD90 and CD105.

Description

Technical field

The invention relates to the acquisition, propagation and characterization of stem cells, in particular to the production of autologous multipotent stem cells from the dermis of a human skin, the propagation of the derived cells and the characterization thereof.

State of the art

One of the most pressing socio-economic problems in developed countries is the general aging of the population. Cardiovascular, autoimmune and neurodegenerative (Alzheimer's, Parkinson's) diseases are among the major health problems associated with aging populations in developed countries. There are no effective treatments for these pathologies, but stem cells can be used to replace damaged cells in the human body. In recent years, stem cell research has shown that with the accumulation of knowledge and the development of practically oriented methods, the introduction of stem cells into medicine is possible. Clinical trials of stem cells to date have yielded promising results in severe pathologies such as myocardial infarction, cardiovascular ischemic diseases, bone and joint aging-related changes, vascular dysfunction, autoimmune diseases, retinopathy, neurodegeneration.

The unique ability of stem cells to transform into specialized cell types is used to "replace" damaged or degenerate cells in the human body, as well as to stimulate functionally defective cells to recover in tissues / organs. Stem cells (autologous stem cells) found in human tissues are the basis for organ homeostasis and self-renewal. According to the definition of stem cells, they have the ability to self-renew during the life of the organ and at the same time to form highly differentiated populations of daughter cells that provide the organ with homeostasis and functionality. Although the number of stem cells in the tissues is small (varying in density from one to 10,000 cells depending on the type of tissue, but generally much lower), they attract a great deal of attention due to their potential applications in biomedicine. Autologous stem cells have significant practical applications, no tissue incompatibilities, and bioethics issues have been reduced to the fact that the cells of the body itself are isolated and, where technically possible, propagated in vitro. Autologous stem cells may be classified according to: the type of tissue from which they are derived; from the process of differentiation of daughter cells; from the set of genes they express. It has recently been discovered that there may be more than one type of stem cell per tissue type. For example, scientific studies suggest that the skin and its derivatives contain at least three intelligent stem cells.

Given that skin is the most accessible and largest human organ (1.5 to 2 m in size for an adult), skin has significant potential for autologous stem cell source application.

A method for growing mesenchymal stem cells is known (WO2007069813 Methods for culturing mesenchymal stem cells and compositions comprising mesenchymal stem cells for repairing skin defects, publ. 21.06.2007). Under this method, subcutaneous fat is used to grow mesenchymal stem cells.

There is a known method for obtaining multipotent stem cells from a human skin derivative, the hair root (WO2006110806 Multipotent adult stem cell, publ. 19.10.2006). This patent application describes a procedure for obtaining and multiplying stem cells, comprising forming a cell suspension, culturing the cells under conditions of multipotent stem cell growth and propagation, and identifying and separating the multipotent stem cells from the culture of culture. The application also discloses cell growth media and growth hormones / additives. An embryonic stem cell (hESC) growth medium consisting of about 80% DMEM / F-12 medium and about 20% embryonic stem cell serum replacement is provided, as well as about 200 nM L-glutamine, about 0.1 mM [betaj- mercaptoethanol, a mixture of about 1% substituted amino acids, and about 4 ng / mL bFGF. The resulting stem cell population was characterized molecularly biologically, and the expression profile of genes characterizing stem cells was added. WO2006110806 describes a method for obtaining stem cells from hair which express the Nanog, Oct4 and Nestin markers and is capable of expressing markers of 9 different cell lines in differentiation experiments, thus confirming their stem potential.

There is a known method for obtaining stem cells from human dermis cells of fibroblasts by dedifferentiating them with cellular environmental additives (US2003185805). The use of the obtained cells to promote the regeneration of damaged tissue is proposed.

There is a known method for extracting stem cells from the roots of the hair on the skin and the dermis of the skin (EP 1718732). Skin stem cells, which are obtained in a known way, are capable of forming skin and hair, as well as other cell types.

A method for isolating multipotent stem cells from an adult mammalian dermis (Jean G. Toma et al., Isolation of a multipotent stem stem from the mammalian skin, Nature Cell Biology, vol. 3, September 2001, pp. 778-784) is known. .

The lack of known techniques is problematic in their practical application to the development of medical methods in accordance with the requirements of European Union regulatory enactments. The main causes are the complex cell acquisition and / or propagation protocols for their implementation in medical technology, as well as the relatively small number of stem cells obtained in methods based on the extraction of cells from hair root tissues. Propagation protocols for cellular medical applications require certain reagents that can be produced in accordance with regulatory requirements, i.e. must be properly certified products and their suppliers. The present invention provides for a simpler and more cost-effective method of cell multiplication with reagents that meet the requirements of clinical use. The present invention makes it possible to obtain autologous stem cells in an amount that is adequate for therapeutic use based on current knowledge and evaluation of clinical results.

Disclosure of the Invention

The present invention relates to the production of autologous multipotent stem cells from human skin dermis, for use in medicine, and to the reproduction of the derived cells and characterization of their quality.

Human skin derivative tissues provide populations of stem cells with multipotential properties - in vitro differentiation potential for neuronal and mesenchymal (osteoblast, chondrocyte and adipocyte) cell formation. The stem cells of each individual skin donor are respectively referred to as autologous multipotent stem cells. The proposed method for obtaining a population of multipotent stem cells includes:

(a) culturing stem cells isolated from the dermis or a portion of an adult human skin under conditions in which multipotent stem cells (at least 10% of the cells) grow and reproduce adherents;

(b) separating non-multipotent stem cells (which enter into suspension). Optimal conditions for growth and multiplication of multipotent stem cells (at least 10% of the cells) are in tissue culture media consisting of DMEM (glucose 4.5 g / L, HEPES 25mM, pyruvate free) or DMEM: F-12 3: 1, with 10% FCS, penicillin and streptomycin (100 u / ml and 100 pg / ml, respectively) with or without 4 ng / ml FGF-2.

Stem cells isolated from the dermis or a part of the skin of an adult human are able to produce cells corresponding to neurons, osteocytes, chondrocytes and adipocytes as a result of differentiation.

The proposed method of characterizing the quality of the resulting autologous multipotent stem cells is characterized in that at least 95% of the stem cells isolated from the dermis or a part of the skin of an adult human being have the following surface markers: CD73, CD90 and CD105. Markers can be traced back to the FACS method. The choice of such markers is recommended by expert reports of the International Society for Stem Celi Reserach (ISSCR).

Multiple potency of autologous multipotent stem cells is evaluated by performing cell differentiation experiments on neuronal and mesenchymal (osteo, chondro, and adipose) tissues. The following positive markers are used: nestin, tubulin bill, GFAP and OC4 (all neurons); FABP4 (adipose); aggrecan (hondro) and osteocalcin (osteo). These markers are determined by immunohistochemistry using specific monoclonal antibodies. In addition, histological staining methods are used: Oil Red O ™ (adipozo) and Alcian Blue ™ (hondro).

The essence of the invention and its implementation are further explained by examples.

Examples of realization of the invention

Example 1:

Autologous multipotent stem cells according to the present invention were obtained using human dermis. Human skin samples (l-2cm ² ) obtained from post-operative material, with the permission of the Latvian Central Ethics Commission, were placed in a 50ml centrifuge tube with sterile PBS solution supplemented with fungizone (0.5pg / ml) and a mixture of penicillin, streptomycin ( 100 µg / ml and 100 µg / ml) and stored at + 4 ° C. After transporting the skin sample to the laboratory, remove the skin sample from the transport solution and place it in a Petri dish. Using a sterile scalpel, cut the skin sample into O, 5x0, 5cm pieces and place in a 15ml tube of dyspase (0.5mg / ml) in PBS solution. The sample is incubated in a disperse solution for 2h at o

C. The laminar sample is removed from the dispassion solution and placed in a Petri plate with PBS solution. Using a sterile tweezers, the epidermis is removed from the dermis. The dermis is cut into small pieces and treated with collagenase XI (50U / ml) in DMEM medium with 10% calf serum supplement for 2-4h at 37 ° C. The epidermis is treated with 0.25% trypsin / EDTA mixture for 2 min at 37 ° C, quenched with 1 ml calf serum. Epidermal cells are centrifuged at 1200 rpm to remove supernatant fluid. Epidermal cells are seeded in 25cm ² cell culture dishes in DMEM / F12 3: 1 medium with 10% calf serum and antibiotics penicillin and streptomycin (100u / ml and 100ng / ml). The dermis treatment reaction mixture is filtered through a 70μ nylon sieve and centrifuged for 10 min at 1500 rpm. The supernatant liquid is aspirated and the precipitate is washed with 10 ml PBS and centrifuged again at 1500 rpm for 5 min. After rinsing, serum tissue culture medium - DMEM / F12 (3: 1) with 10% calf serum and penicillin and streptomycin (100u / ml and 100µg / ml) is added to the cells. When the cells reach 90% confluency, they are trypsinized and prepared for growth - transfer 75 cm in a vial with 10m added! Selected feeds.

.example

Cell trypsinization. The culture medium, trypsin and saline solutions are heated in a water thermostat to 37 ° C. The cell vial is removed from the CO ² incubator. Extract medium from the cell culture vial with a sterile pipette. The cell was rinsed with sterile HBSS (T 25 cm ² or 5 mL; T 75 cm ² or 10 mL). Absorb HBSS and introduce 0.25% trypsin-EDTA in a volume of T 25 cm ² - 1 ml, T 75 cm ² - 3 ml. Close the vial and place at 37 ° C for 2 min. A microscope is used to check that the cells have detached from the surface of the vial. Stop the reaction with calf serum by adding the indicated amounts of T 25 cm ³ - 1 ml, T 75 cm ³ : 3 ml. Add sterile HBSS to make a total volume of 10ml. Using a sterile pipette, take mourned logical one drop of the cell suspension are diluted with Tripānzilā dyes 1: 1, then packed into a cell counting chamber (cell number Sedgwick-Rafter slide chamber 25 plots corresponding to lmm ^3). Transfer the cell suspension to 15ml centrifuge tubes and centrifuge for 5min at 1200 rpm. The supernatant fluid is aspirated with a sterile pipette. Dilute the precipitate to the protocol volume per ml. The cell number is calculated using the formula X (cell number imi) ⁼ cell number in 25 fields x 10,000.

Example 3:

In vitro growth of skin stem cells. The cell suspension (after isolation from skin sample or trypsinization) is placed in 25cm ² cell culture dishes and / or 6-well plates in a 37 ° C incubator with 90% humidity and 5% CO ^{2 in} selected medium (transfer 75cm ^{2 in a} vial with 10mL media). ):

in serum medium: DMEM and F12 3: 1 in volume with 10% calf serum and penicillin and streptomycin (100u / ml and 100pg / ml) or in medium FEB - DMEM and F12 in 3: 1 volume with 2% serum substitute B27, penicillin and streptomycin (100u / ml and 100pg / ml) and growth factors: basal fibroblast growth factor (bFGF; 40ng / ml) and epidermal growth factor (EGF, 20ng / ml), or in a volume ratio of 3 to LIF-DMEM and F12: 1 with 2% serum replacement B27, antibiotics and growth factors bFGF (40ng / ml) and EGF (20ng / ml) and leukemia inhibition factor (10ng / ml).

Example 4

Proliferation and immunostaining. Skin samples (l-2cm ² ) obtained from biopsy or postoperative material, as authorized by the Central Ethics Commission of Latvia, are placed in a 50ml centrifuge tube with sterile PBS solution supplemented with fungizone (0.5pg / ml) and a mixture of penicillin, streptomycin (100 µg / ml and 100 µg / ml) and stored at + 4 ° C. Human skin biopsy or plastic surgery removes the subcutaneous fat and connective tissue. The skin is divided into 1-1.5 cm ² pieces and incubated with 50 mg / ml Dispase II (Boehinger Mannheim) in DMEM medium with 20 mM Hepes on ice for 18-22 hours. Tweezers remove epidermis and fix whole piece to formalin pH 7.2 (Sigma) for 2 hours at room temperature. The fixed epidermis layer can be stored in PBS with 0.2% sodium azide in a refrigerator at 4 ° C for up to 8 weeks if cell staining cannot be done faster.

Claims (6)

Claims A method for obtaining a population of multipotent stem cells, characterized in that it comprises: (a) culturing stem cells isolated from an adult human dermis or a portion thereof under conditions in which multipotent stem cells (at least 10% of the cells) grow and propagate adherents; (b) separating cells other than multipotent stem cells. The method according to claim 1, characterized in that the stem cells isolated from 10 adult human skin dermis or parts thereof are cultured in medium containing culture media in DMEM or DMEM and F-12 in a 3: 1 volume ratio with 10% calf serum (FCS) and penicillin and streptomycin (100u / ml and 100pg / ml). ) with or without 4 ng / ml FGF-2. Method according to claim 1 or 2, characterized in that at least 95% of the origin The 15 cells isolated from the dermis or part of the skin of adult human skin have the following surface markers: CD73, CD90 and CD105. A method according to claim 1, characterized in that the stem cells isolated from the dermis or a part of the skin of an adult human are capable of forming neurons as a result of differentiation. 20 matching cells. The method of claim 1, characterized in that the stem cells isolated from the dermis or a portion of the skin of an adult human are capable of producing osteocyte-like cells as a result of differentiation. The method of claim 1, characterized in that the stem cells isolated from the dermis or a portion of the skin of an adult human are capable of forming chondrocyte-like cells as a result of differentiation. The method of claim 1, characterized in that the stem cells isolated from the dermis or a portion of the skin of an adult human are capable of forming adipocyte-like cells as a result of differentiation.