Резюме
Свидко К.М., Дьомін Ю.А. Клінічне обгрунтування застосування кріоконсервованих клітин кордової крові при індукції експериментальної лімбальної недостатності рогівки.
Пошук нових методів лікування ЛНР в сучасній офтальмології є актуальним питанням. Існуючі на даний момент варіанти не є достатньо ефективними, складні в виконанні і пов'язані з використанням донорської тканини. У даній роботі показаний клінічний ефект застосування кріоконсервованих клітин кордової крові для компенсації лімбальної недостатності рогівки в експерименті.
Ключові слова: кріоконсервовані клітини кордової крові, лімбальна недостатність рогівки, клінічний ефект.
Резюме
Свидко Е.Н., Дёмин Ю.А. Клиническое обоснование применения криоконсервированных клеток кордовой крови при индукции экспериментальной лимбальной недостаточности роговицы.
Поиск новых методов лечения ЛНР в современной офтальмологии является актуальным вопросом. Существующие на данный момент варианты не являются достаточно эффективными, трудны в исполнении и связаны с использованием донорской ткани. В данной работе показан клинический эффект применения криоконсервированных клеток кордовой крови для компенсации лимбальной недостаточности роговицы в эксперименте.  
Ключевые слова: криоконсервированные клетки кордовой крови, лимбальная недостаточность роговицы, клинический эффект.
 Summary
Svidko K.M., Demin Yu.A. Clinical substantiation of application of cryopreserved cord blood cells to compensate limbal stem cells deficiency in experiment.
The search for new treatments for CLD in modern ophthalmology is very actual problem. Existing options currently are not sufficiently effective, difficult to perform and is connected with the using of donor tissue. In this paper we showed the clinical effect of cryopreserved cord blood cells for compensation of CLD in the experiment.
Key words: cryopreserved cord blood cells, limbal stem cells deficiency, clinical effect.

Рецензент: д.мед.н., проф. А.М. Петруня

УДК 617.713-092.4.068

¹Институт проблем криобиологии и криомедицины НАН Украины (Харьков)

61015, Харьков, ул. Переяславская 23

Institute for Problems of Cryobiology and Cryomedicine of the National Academy of Sciences of Ukraine

23, Pereyaslavskaya str., Kharkov, Ukraine 61015

²Харьковская медицинская академия последипломного образования

61176, г. Харьков, ул.Корчагинцев, 58

Kharkov medical academy of Postgraduate Education

61176, Kharkov, 58 str.Korchahyntsev

v.teryshin_lsmu@mail.ru

Introduction.  Loss of vision instead of various diseases of the cornea significantly affects the quality of human life and it is a global social and economic problem. Surgical techniques such as limbal auto- or allografts, amniotic membrane and conjuctiva transplantation in cases are mostly unavailable and require specific skills, the bank of grafts is also required. CCBSC therapy advantage is its lower immunogenicity and availability, as well as possible storage in cryobank.

Purpose - prove CCBC clinical efficacy as a corrector of the CLD in experiment.

Materials and Methods. CCBC  was obtained from maternal end of the umbilical cord after its separation from healthy women. Informed consent was obtained. CBC suspension was cryopreserved in disposable plastic tubes using a two-stage program in high- dextran solution ("Poliglyukin", "Yuri Farm", Ukraine) [11]. The samples were stored in a low- temperature bank of IPCC of NAS of Ukraine. On the day of experiment, CCBC warmed in a water bath at a temperature of 40-41°C. Nonviable cells were prepared by boiling in vitro for 5 minutes. Population structure of CBC was examined by flow cytometry («FACS Calibur», «BD Biosciences», USA) with program Win MDI using monoclonal antibodies to CD3 molecules , CD4, CD8, CD14, CD19, CD34, CD56 («BD», USA).

Eyes of 76 male Chinchilla rabbits weighing 2.0-2.5 kg using CLD model with mitomycin С was studied. CCBC in a dose of 0.5х106 was introduced into each of 8 main limbal zone meridians. Clinical and morphological studies were analyzed to 2, 3, 7, 14 days. Observation of the corneal surface was carried out using the focal lighting, biomicroscopy eye slit lamp Topcon SL-D7. Inflammatory response extent and neovascularization, turbidity intensity, the stroma defect area and corneal opacity degree were assessed.

 Animals were taken out from the experiment under anesthesia by air emboli through the ear vein. The studies were conducted according to the "common ethical principles of animal experimentation" (29/09/01), which is connected with the position of the "European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes" (Strasbourg, 1985).

All the animals were divided into 5 groups: 1- eyes with CLD and CCBC introduction; 2 – CLD model with no treatment; 3 – CLD model with physiological solution; 4 - eyes with CLD and inactivate CCBC introduction; 5 – intact cornea with CCBC transplantation.

Results. In clinical study of eye anterior segment no signs of any specific toxico-allergic response on CCBC suspension use (eyelid dermatitis, chemosis, allergic infiltrates in limbal and perilimbal zone of the cornea) were found. On the 3rd day in groups 2, 3, 4 inflammatory response was evaluated in 2 points. Visually mucous discharge, mixed injection of the conjunctiva, cornea perifocal edema was observed.  Diameter of the epithelial defect in these groups was about 6mm. In group 1, the inflammatory response was less and estimated in 1-2 points, epithelial defect was about 3mm.

Fig. 1. Photo of the cornea on the 3^rd day of experiment.   Inflammatory response in the different groups (a – group 2, b – group 3, c – group 4, d – group 1).

On the 7th day inflammatory response in groups 3, 4 was estimated in 1-2 points, in the group 1 it was estimated in 1 point. During this period corneal clouding was performed. In group 5 cornea retained its transparency and no inflammatory reaction was observed.

On the 14th day inflammatory response in all groups was estimated in 0 points, a persistent corneal opacity in the groups 2, 4 was formed. In group 5 cornea retained its transparency and no inflammatory reaction was observed. In the group 3 was also noted corneal opacity, but it was less than in groups 2, 5. In group 1 the best results were obtained.

Fig. 2. Photo of the cornea on the 14^th day of experiment. Inflammatory response in the different groups (a – group 2, b – group 3, c – group 4, d – group 1).

Conclusions. Maximal inflammatory response and subsequent pronounced corneal opacity developed in groups with experimental CLD and without treatment using physiological saline. The best result was achieved in the group 1. Complete recovery of corneal anterior epithelium to the 14th day was noted, which was due to migration and differentiation of introduced CCBC as well as possible stimulation of own limbal stem cells by them. Achieved clinical effect is a reason to use CCBC for CLD treatment.

Resume. The search for new treatments for CLD in modern ophthalmology is very actual problem. Existing options currently are not sufficiently effective, difficult to perform and is connected with the using of donor tissue. In this paper we showed the clinical effect of CCBC for compensation of CLD in the experiment.

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