RU2016131784A - Способ получения смеси для распыления клеток-фибробластов кератиноцитов человека в растворе и ее применение в качестве регенеративного агента при повреждениях кожи - Google Patents
Способ получения смеси для распыления клеток-фибробластов кератиноцитов человека в растворе и ее применение в качестве регенеративного агента при повреждениях кожи Download PDFInfo
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Claims (19)
1. Способ получения смеси для распыления клеток-фибробластов и кератиноцитов, включающий стадии:
a) разъединение слоя дермы и слоя эпидермиса;
b) инкубирование слоя эпидермиса для получения трипсинизированного эпидермиса;
c) дезинтеграция слоя дермы и пролиферация фибробластов;
d) разъединение слоя эпидермиса;
e) пролиферация кератиноцитов в суспензии;
f) приготовление раствора сшивающего реагента раствора клеточного распыления; и
g) приготовление раствора клеточного распыления, причем на стадии а) получают образец кожи от пациента в стерильных условиях с использованием дерматома для получения листа кожи; промывают этот кожный лист как минимум 3 раза натрий-фосфатным раствором (PBS) 1X, содержащим 10% антибиотика/антимикотика в объемном отношении; промывают как минимум 3 раза в PBS 1X раствора, содержащего 1% в объемном отношении антибиотика/антимикотика; инкубируют с протеолитическим ферментом при концентрации 1,79 ед/мг при температуре 4°С в течение как минимум 16 часов для получения слоя эпидермиса и слоя дермы; по истечении 16 часов инкубирования, разделяют два слоя с использованием стерильного пинцета и размещают их в две разные стерильные емкости.
2. Способ получения смеси для распыления клеток-фибробластов и кератиноцитов по п. 1, отличающийся тем, что кожный лист имеет размер 0,015 дюймов.
3. Способ получения смеси для распыления клеток-фибробластов и кератиноцитов по п. 1, отличающийся тем, что антибиотиком/антимикотиком является пенициллин/стрептомицин.
4. Способ получения смеси для распыления клеток-фибробластов и кератиноцитов по п. 1, отличающийся тем, что в качестве энзима используется диспаза.
5. Способ получения смеси для распыления клеток-фибробластов и кератиноцитов по п. 1, отличающийся тем, что стадия b) включает разъединение и промывание слоя эпидермиса в отдельной емкости как минимум 3 раза раствором PBS 1X и затем инкубирование слоя эпидермиса в 0,025% растворе трипсина в конечном объеме этилендиаминтетрауксусной кислоты в течение 20 минут при температуре 37°С для получения трипсинизированного эпидермиса.
6. Способ получения смеси для распыления клеток-фибробластов и кератиноцитов по п. 1, отличающийся тем, что стадия с) включает размещение слоя дермы в контейнере и механическое дезинтегрирование с помощью инструмента постоянного импульса в стерильных условиях для получения кусочков дермы, размер которых не должен быть более 1 мм × 1 мм, а затем инкубирование в течение как минимум 10 минут, и добавление к стенкам контейнера 33,33% от размера контейнера питательной среды для фибробласта и предотвращение движения кусочков дермы, затем инкубирование при 37°С, 95% RH, и 5% CO2 и 20% О2 для получения монослоя фибробластов.
7. Способ получения смеси для распыления клеток-фибробластов и кератиноцитов по п. 1, отличающийся тем, что стадия d) включает нейтрализацию трипсинизированной эпидермы 50% питательной среды человеческих кератиноцитов, содержащей 10% фетальной бычьей сыворотки, и интенсивное перемешивание в течение как минимум 30 секунд для получения раствора клеток, затем фильтрование раствора клеток через фильтр из нитроцеллюлозы для получения человеческих кератиноцитов в суспензии в отфильтрованном продукте.
8. Способ получения смеси для распыления клеток-фибробластов и кератиноцитов по п. 1, отличающийся тем, что стадия е) включает посев монослоя кератиноцитов в асептических условиях в контейнере с питательной средой и инкубирование при 37°С, 95% относительной влажности, 5% СО2 и 20% О2 в течение периода от 2 до 4 недель, и смену питательной среды до достижения 100% конфлюэнтности кератиноцита.
9. Способ получения смеси для распыления клеток-фибробластов и кератиноцитов по п. 1, отличающийся тем, что стадия f) включает генерирование в асептических условиях 15 мл от 27% в объемном отношении бычьего легочного тромбина при концентрации 11 ед/мл в хлористом натрии 0,9% в объемном отношении и наполнение шприца 15 мл сшивающим реактивным агентом для получения сшивающего реактивного агента раствора клеточного распыления.
10. Способ получения смеси для распыления клеток-фибробластов и кератиноцитов по п. 1, отличающийся тем, что стадия g) включает вращение при 400g в течение 5 минут 50сс аутологичной периферической крови для получения 15% в объемном отношении сыворотки безклеточной плазмы и затем ресуспензирование с 10×106 фибробластов в монослое, и зарядить 15 мл раствора клеточного распыления в 15сс шприце из 15сс, и затем смешивание соединения клеточного распыления с раствором клеточного распыления в распылителе для получения смеси клеточного распыления для фибробластов и кератиноцитов.
11. Способ получения смеси для распыления клеток-фибробластов и кератиноцитов по п. 10, отличающийся тем, что аутологичная периферическая кровь является человеческими тромбоцитами или криоконсервированными тромбоцитами.
12. Способ получения лекарственного средства для лечения поражений кожи, включающий нанесение смеси, полученной способом, описаным в п. 1.
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PCT/MX2015/000001 WO2015119491A1 (es) | 2014-02-04 | 2015-01-15 | Proceso de obtencion de un compuesto de aspersion celular de fibroblastos y queratinocitos humanos en solucion y su uso como agente regenerativo de lesiones cutáneas |
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KR20160132007A (ko) | 2016-11-16 |
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US20160271299A1 (en) | 2016-09-22 |
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EP3103865A4 (en) | 2017-10-25 |
AU2015214649A1 (en) | 2016-08-11 |
HK1232250A1 (zh) | 2018-01-05 |
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RU2016131784A3 (ru) | 2018-10-16 |
CA2938041A1 (en) | 2015-08-13 |
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