Federalnoe gosudarstvennoe byudzhetnoe uchrezhdenie "Natsionalnyj meditsinskij issledovatelskij tsentr transplantologii i iskusstvennykh organov imeni akademika V.I. Shumakova" Ministerstva zdravookhr
发明人:
Sevastyanov Viktor Ivanovich (RU),Севастьянов Виктор Иванович (RU),Basok Yuliya Borisovna (RU),Басок Юлия Борисовна (RU),Nemets Evgenij Abramovich (RU),Немец Евгений Абрамович (RU),Kirsanova Lyudmila
申请号：
RU2019115236
公开号：
RU0002716577C1
申请日:
2019.05.17
申请国别(地区):
RU
年份:
2020
代理人:
摘要：
FIELD: biotechnology.SUBSTANCE: invention refers to medicine, namely biotechnology. Method involves chopping articular porcine cartilage with fragments of size of not more than 0.5 cm, milling and separating particles with size from 100 mcm to 250 mcm. Then decellularisation is performed: at least 3 cycles of cooling at temperature of -196 °C for one hour with subsequent thawing at 35–40 °C for one hour, incubation in three shifts of phosphate buffer (138 mM NaCl, 2.67 mM KCl, 1.47 mM KH2PO4, 8.1 mM Na2HPO4, pH 7.4 (FBS), pH = 7.4), volume 20–500 ml, containing 0.1 % sodium dodecyl sulphate and increasing concentration of Triton X100 (1, 2 and 3 %, respectively), incubation for 12–48 hours at temperature of 37 °C in 1–10 ml of solution containing 30–50 U/ml of DNase Type I in a buffer solution prepared from 10 mM Tris-HCl, 2.5 mM MgCl2, 0.5 mM CaCl2 , distilled water up to 1 l with pH 7.6. Further, cartilage particles are washed by rinsing with 100–500 ml bidistilled water, exposure for 20–500 ml of bidistilled water during 24 hours at room temperature and periodical stirring at rate of 10–500 rpm for one hour three times throughout the day, exposure for at least 48 hours at room temperature in bidistilled water containing ampicillin in amount of 10–20 mcg/ml and amphotericin at rate of 1.5–2.0 mcg/ml, rinsing in 20–500 ml bidistilled water. Thereafter, the cartilage particles are dried and sterilized by γ irradiation in dose of 1.5 Mrad to obtain samples of the desired matrix.EFFECT: invention improves completeness of cell removal due to micronisation and proposed original physical effect (freezing/thawing), chemical (mixture of ionic and non-ionic surfactants) and biological (DNase) factors used in optimal combination, easier cellcellularisation of the matrix by cells by increasing the area for settling while maintaining volume and simplifying observation thereof by increasing the area of the matrix adhered surfaces by preliminary micronisation thereof.3 cl, 7 dwg
