Materials and methods

The experiments in animals were performed according to the General ethical principles of experiments in animals, approved by the 3rd National congress on bioethics (Kiev, 2007) and agreed with the statements of the European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes (Strasbourg, 1985). Thyrocytes and follicles were isolated from thyroid glands of newborn piglets by 3-stage enzyme disaggregation using 1 µg/ml collagenase of type IA and 0.15 µg/ml deoxyribonuclease (Sigma, USA) at 37 C with further cooling and 3-fold washing with the solution, containing 0.2% of bovine serum albumin. Follicular and cellular fractions were separated by filtration through the membrane with of 30 mm pores (Consalt TS, Italy). After filtration the follicular fraction was mainly represented by middle size follicles (> 30 mm) and cellular fraction included thyrocytes, erythrocytes and small follicles (< 30 mm). The obtained fractions with concentration of cells 1× 106 and follicles 1× 105 were cultured in cultural flasks (Sarstedt, USA) with the medium 199, enriched by 10% fetal calf serum (FCS)(Sigma, USA) supplemented with antibiotics as penicillin (100 units/ml, kanamycine (200 µg/ml) and amphotericin B (1 µg/ml) at 37 C in the atmosphere of 5% CO 2 for 6 days. The culture was disaggregated by incubation in 0.25% trypsin mixed with Versene solution in the 1: 1 ratio at 37 C for 3 min with further adding of nutrition medium 199 with 5% FBS, and removed from the substrate by pipeting till the moment of monolayer detaching and then centrifuged at 1,000 rpm. The samples were cryopreserved in the programable freezer (Cryoson 115, Germany) using the cooling rate of 1 degree/min [2] in presence of 10 and 15% DMSO both with and without adding 25% FCS. Thawing was carried-out on water bath at 37 C till

Известно, что эндокринные ткани высоко чувствительны к различного рода ишемическим факторам, осмотическим изменениям, действию низких температур. В литературе широко представлены способы криоконсервирования клеток и ткани некоторых эндокринных органов с использованием низких скоростей охлаждения, в частности 1 градус/мин, и растворов криопротектора ДМСО, который в определенной концентрации обладает выраженным противоишемическим действием [14]. Описаны положительные результаты при замораживании островков поджелудочной железы в присутствии 20%-го раствора ДМСО [12, 13], суспензии клеток надпочечников новорожденных поросят–7%-го [8], суспензии клеток Сертоли фетальных семенников крыс–10%-го [10], органотипической культуры щитовидной железы новорожденных поросят–7%-го [4].