Method for local wound healing by means of biological dressing containing live cells of human diploid fibroblasts

FIELD: medicine.

SUBSTANCE: method for local wound healing involving using a biological dressing which is applied on a wound surface. The biological dressing contains a polymer base of a hydrophobic perforated silicone film coated with a layer of human collagen type I and human diploid cells. The biological dressing is square-shaped and contains the diploid cells in the form of the characterised live cells of M-20 human fibroblasts at the level of passages No. 20-33 in the form of cell monolayers of 70-80% saturation density prepared at a starting density of (4-5)×104 cells per 1 cm2 of the dressing and culturing in a nutrient medium with fibrinolytically active plasma added. Vast injuries may require placing a desired number of the line-on-line dressings on the wound surface. Preferentially, the dressing is applied from the 1st-2nd post-injury day.

EFFECT: improving repair processed in the wound and reducing time of healing.

4 cl, 4 ex, 1 tbl, 3 dwg

 

The invention relates to medicine, and biotechnology.

The modern level of development of biology and medicine enables you to create wound dressings containing diploid cells that can actively influence on the process of wound healing [DS. Sarkisov, A.A. Alekseev, V.P. Fogs in kN.: New methods of treatment of burns using cultured alloparental. International Symposium. Saratov, 1998, p.31]. This approach opens the way to improve efficiency, in particular, local edge treatment of burn wounds [S.V. Smirnov, V.B. have been Khvatov. Innovative technologies for the local treatment of burns in the research Institute of emergency care named. N.V. Sklifosovsky. In the book: the New economy. Innovative portrait of Russia. M, the Center for strategic partnership, 2009, s-390].

The prototype. RF patent for the invention №2373944., 23.06.2008 (Method of treatment of burn wounds. Ermolov A.S., Smirnov ST., Grips V.B. have been, Mironov L.L., Conosco I., Zhirkov E.A., Bocharova B.C.). The method of local treatment of burn wounds includes toilet wound overlay on the surface of a biological dressing consisting of hydrophobic perforated silicone film "Carboxyl P"covered

a layer of human collagen type 1, and as allogeneic fibroblasts characterized using cell lines of diploid human fibroblasts M-22 at the level of 15-25 passages, and run what I used with the first two days after burn injury. Local treatment of burn wounds has led to rapid restoration of the epithelium over the entire surface wounds II-IIIA with good functional and cosmetic result. However, used a strain of diploid human fibroblasts M-22 is very demanding and demanding in terms of cultivation, has a lack of proliferative activity that lengthens the time of manufacture of biological dressings and increases the flow cell. The result is the need for cell lines, but is also suitable for the local treatment of pressure sores, bite wounds, long-term healing and burn wounds. For local treatment of skin lesions is proposed to use diploid human cells - fibroblasts line M-20.

Here is a comparative description of the properties of the line M-20 and a licensed line M-22 (used in the prototype) (table).

Comparative characteristic lines of diploid human cells M-20 and M22
DescriptionM-20M-frooti)
Species, clothMan, the skin and muscles a 10-week embryo, obtained by performing an abortion on medical until the to be. Man, the skin and muscles of 8-week embryo, obtained by performing an abortion for medical reasons.
Where and when selectedIPWA them. M. Lomakov RAMS, 1986IPWA them. M.P. Chumakov, RAMS, 1986
Characteristics of donorCancer, sexually transmitted diseases, hepatitis, tuberculosis in history is not detected; genetic and congenital diseases in the family is not found.
Cultural property
The type of growthfibroblast-likefibroblast-like
Life cycle
Life cycle phaseThe formation of 1-3 passages, active growth 4-40, aging from 41 to 48-52.The formation of 1-3 passages, active growth 4-39, aging 40-70.
The method of cultivationStationary, inoculation 2 times a week during the active growth phase with a sieving coefficient of 1:2-1:3.
Conditions of cultivationEnvironment DMEM with the-glutamine with addition of 10% fibrinoliticeski active plasma (FAP) at 37°C and the content of CO 2-5%.Wednesday Needle MEM with α-glutamine and 10% fetal bovine serum at 37°C and the content of CO2-5%.
Separation of cells from the surface growthA mixture of 0.25% trypsin solution.
Conditions of cryopreservationThe composition createsite environment: DMSO 10%, fetal serum of cattle 40%, medium Needle MEM 50%
Mode cryopreservationThe starting temperature of 20°C, air temperature drops to +4°C at a rate of 1 deg./min; exposure for 10 minutes, reduce temperature to -30 deg./min with a speed of 1 deg./min; extract 10 minutes; reduce temperature to -150°C at a rate of 10 deg./min. Transfer of frozen samples in liquid nitrogen.
Recovery and viability of cells after storage inIn a water bath at 37°C. Viability: not less than 75±5% of cells. Media for cultivation of the same.

Continuation
Karyological properties.Chariot the p person 2n=46,XY Diploid cells 93,3-to 96.9%. Polyploid cells is not more than 1.6%. The strain has a high genetic stability.The human karyotype 2n=46,XY Diploid cells 89,7-93,6%). Polyploid cells is not more than 2.3%.
Isoenzyme featureThe number of bands isoenzymes MR. FDG and LDH and their electrophoretic mobility coincide with those for human erythrocytes. Mr. FDG slow type
Control of contamination:
BacteriaNegativeNegative
MushroomsNegativeNegative
MycoplasmasNegativeNegative
VirusesNegativeNegative
Control of carcinogenic activity
In vivoNot foundNot found
In vitroNot foundSensitivity to virusesPoliovirus strains of A. Sabina 1, 2, 3 types a virus vesicularpustular (air force).The poliovirus strains of A. Sabina 1, 2, 3 types, ECHO, measles, EC, LHM.
Biochemical, genetic markersHLA markers. A*02,03; B*07,40; C*03, 07; DRB1*15, 16; DQB1*05, 06Not determined.
ScopeThe test system for determination of interferon status, to obtain a cellular product designed for the treatment of burn (II-IIIa degree) and long-term healing of wounds.For making polio vaccines from strains of A. Sabina three types.

In addition, it is shown that the cell line M-20 of 20 arcade produce mRNA α-interferon (Ifnα) and interleukins: IL 1β, 2, 4, 6, 8, 10, 18, that explains the mechanism of improvement of reparative processes in wound-healing.

Passport line of diploid human cells M-20

Designation line: M-20; Species: human; line established in 1986 in the state of IPVA them. M.P. Chumakov RAMS

Cultural properties:

1. The type of growth: fibroblast-like

2. The number of passages: 50±2

3. Phases of growth: I-1-3 passages, II-4-40, II-41-(50±2)

4. The method of cultivation: stationary, inoculation 2 times a week

5. The cultivation conditions: environment DMEM with α-glutamine with addition of 10% fibrinoliticeski active plasma (FAP) at 37°C and 5% CO2in the atmosphere.

6. Method of separating cells from the surface growth: a mixture of 0.25% solution of trypsin and 0.02% solution of Versene in the ratio (1:1)

Storage conditions: at a temperature of minus 196°C (in liquid nitrogen); (a) composition createsite environment: environment DMEM-50%, cryoprotector DMSO-10%, serum embryos cows - 40%. b) freezing: the starting temperature of 20°C, air temperature drops to +4°C at a rate of 1 deg./min; exposure for 10 minutes, reduce temperature to -30 deg./min with a speed of 1 deg./min; extract 10 minutes; reduce temperature to -150°C at a rate of 10 deg./min. Transfer of frozen samples in liquid nitrogen.

Recovery and viability of cells after cryo-storage:

in a water bath at 37°C. Viability: not less than 75±5% of cells. Media for cultivation of the same.

Isoenzyme description: Number of strips of isoenzymes MR. FDG and LDH and their electrophoretic mobility coincide with those for human erythrocytes. Mr. FDG slow type.

Karyological characteristics: human Karyotype 2n=46, XY. Diploid cells 93,3-to 96.9%. Polyploid cells is not more than 1.6%. The strain has a high gene is political stability.

Control of contamination: bacteria, fungi, mycoplasmas when planting on a selective nutrient medium, the infection of chick embryos and by electron microscopy study revealed that viruses have been detected in animal studies, cell cultures, in chicken embryos, by means of electron microscopy.

Oncogenic security: in vivo - in the study using linear CBA mice tumors were not formed; in vitro control by reverse transcriptase negative.

Biochemical, genetic markers: a) HLA - markers: A*02, 03; B*07, 40; C*03, 07; DRB1*15, 16; DQB1*05, 06. b) Cell line M-20 of 20 arcade produce mRNA α-interferon (Ifnα) and interleukins: IL 1β, 2, 4, 6, 8.10, 18.

Thus, the line of diploid human cells M-20 is characterized according to the methodical recommendations "Certification transplantable cell lines substrates production and control of medical immunobiological preparations RD-42-28-10-89. THE USSR MINISTRY OF HEALTH. M., 1989.- P.16]. It is characterized on the security in accordance with who recommendations and the requirements of Gneissic mibp them. L.A. Tarasevich. In IPVA them. M.P. Chumakov RAMS have seed banks and working cell, capable of providing all the needs of production and research. Line M-20 has a higher proliferative activity than the line M-22, due to the COI is whether under cultivation of cells FAP.

These findings provide the rationale for use of these cells in a new biological dressing to accelerate the healing process in the treatment of bedsores, burns, bite and long-term healing of wounds.

Technical solution aimed at creating a biological dressing with improved properties due to the use of human fibroblasts line M-20, with a higher proliferative activity in the cultivation with the addition of fibrinoliticeski active plasma (FAP), which provides an improvement of reparative processes in the wound and reduce healing time. This armband is made in large cups square shape that provides an additional advantage is the possibility of covering the wound surfaces, including by placing the required number of bands "butt" on the wound field that allows you to close all wound field with extensive damage.

Thus, the object of the invention is a biological dressing for the local treatment of wounds containing polymer base of the hydrophobic perforated silicon film coated with human collagen type I, and diploid human cells, which as a diploid cell contains characterized by living cells fibroblasts line M-20 at the level of p is Saga No. 20-33 in the form of a monolayer of cells to 70-80% of the density of saturation, obtained when the initial density of planting (4-5)×104cells on 1 cm2headbands and cultivation in a nutrient medium with the addition of fibrinoliticeski active plasma (FAP), this armband has a square shape.

The object of the invention is also a method of local treatment of wounds which includes applying to the surface of the wound biological dressings containing polymer base of the hydrophobic perforated silicon film coated with human collagen type I, and diploid human cells, in which a biological dressing is made in the form of "square" and contains as diploid cells characterized by living cells fibroblasts line M-20 level passages No. 20-33 in the form of a monolayer of cells to 70-80% of the density of saturation obtained at initial density of planting (4-5)×104cells on 1 cm2headbands and cultivation in a nutrient medium with the addition of the FAP. When extensive damage can accommodate the required number of bands "butt" on the wound field. Preferably, the bandage should be used 1-2 days after injury.

Example 1. An example of manufacturing a biological dressing.

At the bottom of a square Petri dishes (12×12 cm) with an area of 144 cm2(in the prototype used a round Cup with a diameter of 9 cm and an area of 63 cm2) put hydrophobic, transparent, perforin the bathroom silicone film "Carboxyl-P, cover with a layer of collagen type 1 human, dried, subjected to sterilization by gamma-rays. In a sterile room in the prepared bowl Petri placed a suspension of fibroblasts line M-20 (20-33 passage), seeding of cells is (4-5)×104cells on 1 cm2. Cells cultured overnight in CO2-incubator at 37°C in atmosphere containing 5% CO2. Use a nutrient medium, DMEM with α-glutamine for cell cultures containing 10% FAP. After 18-24 hours, the cells form a monolayer 70-80% density saturation. The thus prepared biological bandage is taken to the clinic in a sealed box (Fig.1, 2).

Figure 1. Biological bandage on the basis of cultivated alloparental line M-20 (and. appearance bandages, century scheme bandages, S. cell line M-20).

Figure 2. Biological dressing containing fibroblast line M-20 before application to the wound.

Example 2. The imposition of a biological dressing in the example of burn wounds: the wound surface to remove dead tissue, products of combustion and pollution. On the prepared wound impose a biological dressing cells (fibroblasts M-20) paying to the surface of the wound. Wound covering fixed wet-drying gauze bandage. The bandage should be used 1-2 days after burn injury. Following ligation are performed every 2-3 days. Transparent PVC the durability of silicone polymer base allows you to monitor the condition of the wound and to monitor its healing, and the gas permeability of the film is comparable to that of healthy skin, provides metabolic comfort in the wound. The hydrophobic nature of the film enables an easy, painless removal in ligation without damage formed on the surface of the wound regenerierung layer neoepitope.

Examples 3-4. Clinical examples of the use of biological dressings containing fibroblasts line M-20 (figures 3 and. - example 3 - example 4):

Example 3. Patient, 32 years old, diagnosis: flame burn I-II-IIIA extent of 65% of the body surface. After 18 hours after injury produced by primary surgical treatment of the wound and the area of 2260 cm2imposed 15 biological dressings containing living fibroblast line M-20 (90 million cells). At 4 days after imposition of a biological dressing (80 million cells across the wound surface of the back layer formed of neoepitopes; 6 days in places applying bandages were observed complete epithelialization of the wound surface without signs of hypertrophy of the epidermis. Brushstrokes-prints from the surface neocapitalist testified to the presence of microflora typical for normal skin. To 12 days in formed epithelium were signs of recovery pigmentation and hair.

Example 4. Patient S., 35 years old, diagnosis: flame burn I-II-IIIA extent of 25% of the body surface. After 24 hours post-injury PR is plagued primary surgical treatment of the wound and the area of 1200 cm 2imposed 8 biological dressings containing living fibroblast line M-20 (40 million cells). On the 4th day imposed a biological dressing containing living fibroblasts (40 million cells)on day 7 in places applying bandages were observed complete epithelialization of the wound surface without signs of hypertrophy of the epidermis. To 15 days in formed epithelium were signs of recovery pigmentation and hair.

Monitoring of patients within the next 2-3 months indicates the formation of a full skin with normal elasticity, pigmentation and hair growth. At check in 3-6 months the skin in the application area of alloparental smooth, supple, normal color with hair. The use of biological dressings comprehensively examined cell lines of live diploid fibroblasts M-20 ensures biological safety cell material and provides improved reparative processes in the burn wound. The presence of seed banks and working cell thoroughly characterized cell lines allows timely and fully meet the needs of the clinic.

1. A biological dressing for the local treatment of wounds containing polymer base of the hydrophobic perforated silicone the film, covered with a layer of human collagen type I, and diploid human cells, characterized in that as a diploid cell contains characterized by living cells fibroblasts line M-20 level passages No. 20-33 in the form of a monolayer of cells to 70-80% of the density of saturation obtained at initial density of planting (4-5)×104cells on 1 cm2headbands and cultivation in a nutrient medium with the addition of fibrinoliticeski active plasma (FAP), this armband has a square shape.

2. The method of local treatment of wounds which includes applying to the surface of the wound biological dressings containing polymer base of the hydrophobic perforated silicon film coated with human collagen type I, and diploid human cells, characterized in that a biological dressing is made in the form of "square" and contains as diploid cells characterized by living cells fibroblasts line M-20 level passages No. 20-33 in the form of a monolayer of cells to 70-80% of the density of saturation obtained at initial density of planting (4-5)×104cells on 1 cm2headbands and cultivation in a nutrient medium with the addition of FAP.

3. The method according to claim 2, characterized in that in case of extensive damage, place the required number of bands "butt" on the wound field.

4. The method according to any the of claim 2 to 3, characterized in that the bandage is applied with 1-2 days after injury.



 

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