RU2142820C1 - Allograft and method for repairing skin integument having injuries of various etiology and method for producing the allograft - Google Patents

Abstract

FIELD: medicine. SUBSTANCE: allograft has cells of diploid human embryo lung fibroblast strain covering cellophane film. Method involves treating the film with detergent, washing and degreasing it, and distributing diploid human embryo lung fibroblast cells over its surface. Treatment method involves stimulating boundary and regional epithelialization of the proper tissues. EFFECT: enhanced effectiveness in treating skin wounds due to burns, frostbite, phlegmonous inflammation; accelerated healing and cleaning skin wound surface from pus. 3 cl

Description

 The invention relates to biology and medicine, namely to transplantology.

 In clinical practice, a number of different methods are known for repairing damaged skin using in vitro cultured epidermal skin cells. However, the widespread use of keratinocyte grafts is hindered by quite significant technological disadvantages: the difficulty of keratinocyte cultivation, the need to use expensive nutrient media, growth additives and substrates, the significant delay in the treatment with cellular autograft, the restriction in the use of primary allokeratinocytes due to the impossibility of their sufficient certification, and also a large dependence of the result transplantation from the degree of preparedness of a burn wound.

 The use of an alternative source of transplanted cells — fibroblasts — to restore skin integument provides several advantages. Since fibroblasts, through the synthesis of extracellular matrix components, stimulate both keratinocyte adhesion and their proliferation followed by differentiation, grafts from cultured fibroblasts can be used in a large number of cases where there is no total damage to the skin and stimulation of the proliferation of their own epidermocytes in preserved transdermal foci is possible layers.

 Technical solutions are known in which surface-dependent cell cultures, in particular skin cells, are immobilized on substrates formed from synthetic and natural polymers, as well as on the basis of permeable filters and dressings. The use of these substrates as a substrate for skin cells is promising in terms of facilitating the transfer of cellular material to the wound surface, and also avoids unnecessary trauma to the cells during their enzymatic removal.

 However, the use of the above materials is limited either by their high cost, which especially concerns biological materials, or by their inaccessibility, for example, for newly synthesized materials, or by the great inconvenience of controlling cell growth and the wound healing process, which is associated with the use of opaque filters and dressings.

 Closest to the claimed invention in technological execution and the results obtained is an allograft of allofibroblasts on the membrane to restore the skin of the burned. According to this method, an allofibroblast transplant is obtained by fermentation of a calcined dermis, after which the cells are subjected to 5-7 fold passaging. The last subculture is carried out in Heraus plates. A 3-5 day old monolayer is delivered to the operating room and transplanted with the membrane onto skin wounds.

 Despite the fact that this method has established itself as quite effective in the treatment of burns with various burn depths, as well as in the treatment of long-term non-healing donor wounds, it has some disadvantages. This method involves the use of non-standardized cellular material, which can lead to unpredictable results and difficulties in developing a treatment regimen. The need to work with primary culture, as well as its preliminary certification, increases both the time of preparation of cellular material before transplantation and the required material costs. In addition, the growth of fibroblasts in Heraus plates also leads to a rise in the cost of this method.

 The technical objectives of the invention are the creation of an allograft based on a certified culture of fibroblast cells and cheap and affordable raw materials for the substrate and the development of an effective method of treating skin lesions of various etiologies using the obtained allografts with a simultaneous decrease in labor input and material costs.

 The tasks are solved by obtaining an allograft by cultivating a certified diploid strain of human lung embryo fibroblasts on a substrate of cellophane and gelatin and then transferring the obtained allograft to burn surfaces, long-term non-healing skin wounds or other skin defects.

SUMMARY OF THE INVENTION Allograft. Cells of the diploid strain of human lung fibroblasts L-68 are seeded at a concentration of 80 - 100 • 10 ⁵ cells / cm ² on a cellophane substrate.

Characterization of the diploid strain of the human lung embryo L-68:
1. The name of the strain - L-68
2. Collection code - IMB-141
3. Species - Man
4. Source of tissue - Human embryo lung 11 weeks.

5. Type of growth, life cycle - Fibroblast-like, 40 ± 10
6. Description of the karyotype - 2p = 46 (XX)
7. The presence of marker chromosomes - None
8. The percentage of ploidy - Polyplodia - 22 cells per 1000 metaphases
9. Structural disorders - 25 per 1000 cells
10. Controlling Species Identity - Human Karyotype
11. Contamination by bacteria, fungi, viruses, mycoplasmas - None
Allograft production. To obtain allograft, use the diploid strain of fibroblasts of the human lung embryo L-68 [15], the inoculum and working banks of which were certified by GISK for the production of immunobiological preparations [16].

For cultivation, the ampoule with L-68 cells is thawed in a water bath at a temperature of 37 ^o C, the suspension is transferred to a culture vessel, incubated in a Igla MEM nutrient medium with the addition of 10% cattle fetal serum at a temperature of 37 ^o C and passaged until the required volume is obtained cells. The last subculture is carried out on a cellophane film placed on the bottom of the Petri dishes.

The substrate for cultivation is prepared as follows. The cellophane film is cut to the size of the bottom of the cup, placed in a 7x detergent solution for 2 hours, then washed with running and distilled water, dried, degreased with ethyl alcohol and autoclaved at 1.1 atm for 20 minutes. Under sterile conditions, the substrate is transferred to a Petri dish and treated with a 1% gelatin solution to better attach the film to the bottom of the cup and evenly distribute the cells. A cell suspension is sown on the prepared substrate at a concentration of 80-100 • 10 ⁵ cells / cm ² , the growth medium is added to the desired volume, cultured at 37 ^° C in a CO ₂ incubator for 1-5 days, after which the cups with prepared allografts pass into the dressing room.

The essential features of the invention are:
- use when receiving an allograft as a cell culture of fibroblasts of the human lung embryo L-68, which were not previously used for this purpose; L-68 cells are pre-certified and stored in a sufficient number of ampoules;
- the substrate for the cultivation is a specially treated cellophane film.

 From literature data, it is known that fetal fibroblasts are more effective in stimulating epidermocytes, and, unlike similar adult cells, actively migrate to areas of mechanical damage to the cell layer. A number of authors have also shown that fetal fibroblasts contribute to wound healing without scarring, due to their metabolism. However, as shown in the experiment, due to their metabolism. However, as shown in the experiment, in comparison with fetal fibroblasts of human skin, the proliferation index for L-68 cell culture is 1.5-2.0 times higher, which facilitates the scaling process during cultivation.

 The cellophane film prepared in this way has good adhesive properties, as well as is moisture permeable and non-toxic, which in itself is important both for cell cultivation and for wound management [19]. In addition, the transparency of the film allows you to control both cell growth in vitro, and the state of the wound. An important factor is the cheapness and availability of the material.

 Such allografts were first obtained by the authors, are not known from literary sources, which allows us to talk about the compliance of the technical solution with the criteria of "novelty" and "inventive step".

 A method of restoring the skin. Using tweezers, the prepared allograft is transferred to the patient’s wound with the cells down and covered with an aseptic dressing. For 2-5 days after transplantation, a white “film” appears under the disk on the wound, after which the substrate is removed. On the 5-8th day, wounds are epithelized.

 An essential feature of the invention is the use of an allograft containing cells of the diploid strain of fibroblasts of the human lung embryo L-68, deposited on a cellophane substrate. The cell substrate is applied downward to the wound. After epithelization, the substrate is removed.

 The use for restoration of the skin of the allograft based on fetal fibroblasts of the human lung L-68, cultured on a cellophane film as a substrate, is not known from literature, indicates that the claimed invention meets the criteria of "novelty" and "inventive step".

 The following are examples of the implementation of the invention.

 Example 1. Patient B. (case history No. 1869), with a diagnosis of a burn with a flame of the trunk and extremities of the I-IIIa and IIIb-IV degrees after chemical necrectomy, performed autodermoplasty with a split skin graft taken from the front surface of the right thigh. In the postoperative period, the wounds of the donor sites were healed, with conservative treatment, the tendency to healing was not observed. April 3, 1997, allografts with L-68 cells were transplanted to the wounds of donor sites. On the third day, when removing cellophane disks, wound cleansing from pus was noted, on day 7, the wounds were completely epithelized.

 Example 2. Patient K. (case history N-1155) with a diagnosis of frostbite of fingers and hands of the II-III degree after cleansing the wound of the left hand, where there was a flaccid, slow epithelization, on the wound surface on January 28, 1997 allografts with L cells were transferred -68. On the 3rd day, pronounced edge epithelization was observed, on the 8th day after transplantation, complete epithelization was observed.

 Example 3. Patient S., 20 years old (medical history N-9091), was admitted to the department with a diagnosis of phlegmon of the anterior surface of the right lower leg. A necrectomy operation was performed, as a result of which an 18 x 10 cm leg wound was formed. Despite complex treatment, no tendency to wound healing was noted. After the wound was filled with delicate granulations, on December 31, 1996, an autodermoplasty was performed with a split skin flap, which was lysed in the postoperative period. On January 28, 1997, an allograft with L-68 cells was transplanted onto the wound surface formed after lysis. On the 2nd day, under the disks, especially from the edges of the wound, a pronounced marginal epithelization was observed in the form of a white border 3-4 mm wide. On day 8, the wound was closed with delicate whitish skin. Histological examination confirmed the closure of the wound with epithelium.

Example 4. Patient I., 44 years old (medical history N-1671) with burns of a flame of degrees IIIa and IIIb, transplanted cultured allofibroblasts (L-68 cells on cellophane disks) were transplanted. In areas with IIIa lesion on the 12th day, complete epithelization occurred; in small areas of 15-30 cm ² with IIIb lesion, wound sizes decreased as a result of marginal epithelization compared with the control, for the treatment of which cultured cells were not used.

 Thus, the above examples show that the used allograft and method for restoring the skin are effective in the treatment of skin wounds of various etiologies: burns; donor, formed after the collection of a skin flap during an autodermoplasty operation obtained as a result of frostbite, phlegmonous inflammation. Fibroblasts transplanted on a substrate stimulate marginal and islet epithelization of their own tissues, accelerate the healing process of the skin. Moreover, these examples indicate that transplanted embryonic fibroblasts during wound infection contribute to cleansing the wound surface from pus.

 The L-68 cell culture is available in sufficient quantities, certified and stored in a frozen state. Based on examples of successful treatment of skin lesions, it is logical to say that the inventions meet the criterion of "industrial applicability".

Claims (3)

1. An allograft for restoration of the skin, containing a polymer substrate with human cells applied to it, characterized in that the polymer substrate is made of cellophane, and certified human cells of the diploid strain of fibroblasts of the human lung embryo L-68 at a concentration of 80-100 are used as human cells • 10 ⁶ cells / cm ² per substrate. 2. A method of producing an allograft for restoration of the skin, characterized in that it includes treating the cellophane substrate with a detergent, washing it with distilled water, drying, degreasing with ethyl alcohol, coating with 1% gelatin, applying certified cells of the diploid strain of the human lung embryo L-68 at a concentration of 80 - 100 • 10 ⁵ cells / cm ² substrate, previously grown in suspension, which is cultivated for 1 to 5 days in a growth medium at 37 ^o C and CO ₂ incubator.  3. A method of restoring the skin with injuries of various etiologies by applying an allograft on a polymer substrate with the cells down to the wound, characterized in that the allograft obtained according to claim 2 is covered with an aseptic dressing, removed after epithelization.