RU2212792C2 - Solution for cryopreservation of skin and cornea - Google Patents

Abstract

FIELD: medicine, ophthalmology, dermatoplasty. SUBSTANCE: solution for cryopreservation of skin and cornea includes cryoprotector, medium 199 based upon Hanks' solution and stimulator of cellular division. As cryoprotector the suggested solution contains dimethylsulfoxide and/or propylene glycol, and/or glycerol in concentration ranged 10-90% against medium, but predominantly 10-20%. As stimulator for cellular division the suggested solution contains epidermal growth factor and/or insulin, and as epidermal growth factor it contains mitogen, polypeptide, in particular. Solution is considered to efficient medium for cutaneous cryopreservation that favors to develop single technology for keeping viable allogenous and autologous skin and single bank of cutaneous and corneal transplants. The present innovation provides higher efficiency and decreased cost in treating patients with different cutaneous and corneal lesions. EFFECT: higher efficiency. 4 cl

Description

 The invention relates to medicine, namely to solutions for cryopreservation of the skin and cornea. The invention can be used to create stocks of viable grafts of the skin and cornea in surgery and ophthalmology.

 Viable allogeneic skin can be used to temporarily restore the skin and stimulate reparative processes in the wound. It can also be used as a source for allogeneic keratinocyte and fibroblast transplants, in combination with autodermoplasty, or as a basis for subsequent autologous keratinocyte transplantation. In order to reduce the number of skin sampling operations in patients with burns and permanent restoration of the skin, viable autologous skin can be used.

 Known solutions for storing skin, for example, a solution containing dimethyl sulfoxide, glycerin, formalin, alcohol and distilled water (RU 2144764 C1, 01.27.2000).

 The disadvantages of this method are the relatively low efficiency. This is due, in particular, to cell damage, short shelf life of the skin.

 Closest to the claimed invention in technical essence is a method of cryopreservation of viable cells using a cryoprotectant such as dimethyl sulfoxide, propylene glycol or polyethylene glycol (WO 96/39812 A3, 12.19.1996).

 Common disadvantages of these solutions are the relatively low efficiency of keeping cells alive, high complexity and cost of storage.

 The aim of the invention is to create an effective environment for cryopreservation of the skin, which contributes to the creation of a unified technology for the storage of viable allogeneic and autologous skin, increasing the efficiency and reducing the cost of treating patients with various injuries of the skin and cornea.

 This goal is achieved in that in order to maintain viability and preserve the specific function of cells for a long time, a solution was developed containing 199 medium based on Hanks solution, cryoprotectant and epidermal growth factor.

 Environment 199 was developed in 1950. It is characterized by a wide range of nutrients and their relatively low concentration (GP Pinaev, Methods of cell cultivation. Collection of scientific papers. - L .: Nauka, 1988, p. 56). Medium 199 is currently used without any additives as support for primary cells.

 Hanks's solution is a dissolved phenol red dye in saline (Yasuda Y. et al. Jornal of Reproduction and Fertility, 1992, 96, 521-528).

 Any mitogen of polypeptide nature, in particular, epidermal growth factor, insulin, etc. can be used as a stimulator of cell division.

 Any reagents known for their function can serve as a cryoprotectant, for example, dimethyl sulfoxide, propylene glycol, glycerin, etc. Moreover, the concentration of cryoprotectant can be from 10 to 90% of the medium, and preferably 10-20%.

 The solution is obtained by simple mixing of the components. For example, glycerol 100-150 mg / ml is added to the finished 199 medium solution based on the Hanks solution, homogenized, then mitogen from 7 to 12 ng / ml is added.

After the skin is placed in this solution, it is frozen and stored in freezers at a temperature of -18 ^o C, -70 ^o C or in liquid nitrogen at -196 ^o C. As necessary, skin grafts are thawed and whole or perforated rags are applied to the wounds. After 45 days of storage at -70 ^o With the number of living cells in the skin decreases on average by only 29%, which allows to obtain during the cultivation of keratinocytes (the main type of epidermal cells) colony. The cryopreservation solution is physiologically compatible and does not need to be removed before application to the patient.

 The ability to store viable skin at low temperatures with a view to its subsequent use as an independent coating or to obtain cell transplants indicates the compliance of the proposed solution to the patentability condition of "novelty".

 Using the totality of these features in their functional unity to achieve the task attests to the compliance of the proposed solutions to the condition "inventive step".

 The use of these features of the invention allows to obtain a new technical result - to improve the quality of skin grafts and obtain viable skin similar in properties to normal skin, and create a bank of skin grafts with a long shelf life.

 Example 1. The method was tested and implemented when performing plasticization of a granulating wound in a rat by transplanting an autologous skin flap after storage on a model with the exception of wound contraction.

 Test report 30, rat 12, male.

05/10/99 a full-layer skin flap with an area of 0.7 cm ^{2 was} taken from a Wistar rat weighing 250 g under general anesthesia. The biopsy sample is placed in a glass container with a developed solution. Freezing was carried out at a speed of 1-2 ^o C per minute at -70 ^o C. After 27 days of storage (three days before transplantation), a full-layer skin flap was excised into the region of the upper third of the back. To prevent contraction, a plastic ring with vertical and horizontal surfaces was sewn into the wound. After 30 days of storage, the skin was thawed at a constant temperature of about 42 ^o C, after which it was transferred to a granulating wound. A Jelonel dressing and a tissue dipped in physiological saline with gentamicin (0.16 mg / ml) were placed over the graft.

 When examining the wound after 5 days, the presence of a transplanted flap was noted. The implanted grafts were pale pink in color, adhering to the wound surface. The edges of the flaps could not be separated from the granulation tissue. When a biopsy was taken, intense bleeding occurred at the incision site of the accustomed skin. During histological examination, a partial detachment of the epidermis was observed. No visible changes were found in the dermis. The basement membrane is retained. By the 15th day, the transplants had the same dimensions, closely adjoining the wound. The surface of the flaps was covered with a thin, smooth, shiny epithelium. During histological examination, the underlying granulation tissue had close contact with the dermis of transplanted skin flaps. In the deep layers of the dermis, increased vascularization, leukocyte infiltration and an increase in the number of fibroblasts were observed. In the area of dermo-epidermal contact, there was a violation of the connection of the dermis and epidermis along the entire length of the skin flap, but exfoliation of the epidermis was not observed. The basement membrane of the epidermis was not broken. In the epidermis, the phenomena of acanthosis and hypertrophy of keratinocytes in the granular layer were determined. At the same time, keratogialin granules were absent in enlarged keratinocytes. Subsequently, epithelial migration to granulation tissue and hair growth in the graft were observed from the edges of the transplanted skin. By the 30th day, the surviving flap slightly increased in size due to regional epithelization. The graft had a pale pink color, held tightly to the wound bed. A thin strip of young epithelium was visible on the granulation tissue around the grafts. In the center, active hair growth was observed. During histological examination of skin graft grafts, granulation tissue was absent. Skin graft grafts corresponded to normal skin in structure. In the upper layer of the dermis, the number of fibroblasts is increased. In the area of dermo-epidermal contact, a violation of the connection of the dermis and epidermis was noted in some places, but exfoliation of the epidermis was not observed. The epidermis was thickened due to the middle layers. The keratinization process is not disturbed. No signs of phenotypic transformation of keratinocytes were found.

Example 2. Patient S-va, 69 years old, medical history 14559, was hospitalized in the burn center of the Research Institute of the Joint Venture named after N.V. Sklifosovsky with a diagnosis of a flame burn of II-IIIA, B degree of the trunk, right buttock and right thigh with a total area of 20% of the body surface (PHB-9%). In connection with the patient’s refusal from the operation of autodermoplasty, burn wounds were treated conservatively. As a result of the comprehensive treatment on the 50th day, the patient remained granulating wounds on an area of 1.5% of the body surface. On the 51st day after the injury, the patient underwent allotransplantation of epidermal keratinocyte strata grown from cadaver skin stored in a developed preservative for 20 days. The first ligation was performed after three days, with marked marginal epithelization, as well as the appearance of several small (about 3-5 mm ² ) epithelization islands. The area of wounds decreased by about 1/3, but the cell layer was not visually determined. The wounds were covered by the dressing "Jelonel"; on top - a bandage with levomekol. In subsequent dressings after 6 and 8 days from the moment of transplantation, a progressive reduction of wounds due to regional epithelization and an increase in the area of epithelialization islands was noted. By the 10th day, the patient had wounds in an area of about 3 cm ² . On the 15th day after transplantation (66th day after the injury), the skin was completely restored.

 The proposed composition ensures the preservation of the viability of the skin and cornea for a long time and allows you to create a bank of transplants suitable for closing wounds and burns of the skin and cornea.

Claims (5)

 1. A solution for cryopreservation of the skin and cornea, including a cryoprotectant, characterized in that the solution additionally contains 199 medium based on Hanks solution and a cell division stimulator.  2. The solution according to claim 1, characterized in that the solution contains dimethyl sulfoxide and / or propylene glycol and / or glycerol in a concentration of from 10 to 90% to the medium, and preferably from 10 to 20%, as a cryoprotectant.  3. The solution according to paragraphs. 1 and 2, characterized in that as a stimulator of cell division, the solution contains epidermal growth factor and / or insulin.  4. The solution according to paragraphs. 1.3, characterized in that as an epidermal growth factor, the solution contains mitogen.  5. The solution according to paragraphs. 1 and 2, characterized in that the solution contains a polypeptide as a mitogen.