Альгинат гидрогелей для трехмерных органной культуры яичников и Яйцеводы
Summary
Культура нормальных клеток в трехмерном контексте представляет альтернативный метод для изучения ранних событий, необходимых для сотовых трансформации и опухолей. Этот метод используется для выращивания нормального яичника и яйцевода клетках для изучения ранних событий в яичниках образование рака.
Abstract
Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ovarian cancers is still in question and might be either the ovarian surface epithelium (OSE) or the distal epithelium of the fallopian tube fimbriae2,3. Culturing the normal cells as a primary culture in vitro will enable scientists to model specific changes that might lead to ovarian cancer in the distinct epithelium, thereby definitively determining the cell type of origin. This will allow development of more accurate biomarkers, animal models with tissue-specific gene changes, and better prevention strategies targeted to this disease.
Maintaining normal cells in alginate hydrogels promotes short term in vitro culture of cells in their three-dimensional context and permits introduction of plasmid DNA, siRNA, and small molecules. By culturing organs in pieces that are derived from strategic cuts using a scalpel, several cultures from a single organ can be generated, increasing the number of experiments from a single animal. These cuts model aspects of ovulation leading to proliferation of the OSE, which is associated with ovarian cancer formation. Cell types such as the OSE that do not grow well on plastic surfaces can be cultured using this method and facilitate investigation into normal cellular processes or the earliest events in cancer formation4.
Alginate hydrogels can be used to support the growth of many types of tissues5. Alginate is a linear polysaccharide composed of repeating units of β-D-mannuronic acid and α-L-guluronic acid that can be crosslinked with calcium ions, resulting in a gentle gelling action that does not damage tissues6,7. Like other three-dimensional cell culture matrices such as Matrigel, alginate provides mechanical support for tissues; however, proteins are not reactive with the alginate matrix, and therefore alginate functions as a synthetic extracellular matrix that does not initiate cell signaling5. The alginate hydrogel floats in standard cell culture medium and supports the architecture of the tissue growth in vitro.
A method is presented for the preparation, separation, and embedding of ovarian and oviductal organ pieces into alginate hydrogels, which can be maintained in culture for up to two weeks. The enzymatic release of cells for analysis of proteins and RNA samples from the organ culture is also described. Finally, the growth of primary cell types is possible without genetic immortalization from mice and permits investigators to use knockout and transgenic mice.
Protocol
Discussion
Развитие трехмерной органной культуры система, учитывающая альгинат гидрогелей представляет универсальный метод для анализа различных типов тканей от самых разнообразных организмов. Использование 3D-культур может быть распространено на области тканевой инженерии и регенеративной ?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
Эта работа была поддержана исследования рака яичников фонд [грант LT/UIC/01.2011], МСЖД центр клинической и поступательного науки, МСЖД онкологический центр, Национальный институт здоровья грант C06RR15482.
Эксперименты на животных были проведены в соответствии с руководящими принципами и правилами установленными AAALAC рамках утвержденных по уходу за животными протоколов уходу и использованию животных комитетом на МСЖД. Животные для этого проекта размещены в барьер номеров в биологических ресурсов лаборатории (BRL) в университете штата Иллинойс в Чикаго. BRL имеет полностью ветеринарного персонала, который контролирует животных по крайней мере два раза в день и дает рекомендации по уходу за животными.
Materials
| Name of the reagent | Company | Catalogue number | Comments (optional) |
|---|---|---|---|
| Leibovitz L-15 medium | Gibco | 11415 | |
| αMEM medium | Gibco | 12000-022 | |
| Sodium alginate | Novamatrix | ||
| Sodium cacodylate | Sigma Aldrich | C0250 | |
| Polypropylene mesh fiber | McMaster Carr | 45″ wide roll; 0.0041″ opening | |
| Lipofectamine 2000 | Invitrogen | 11668 | |
| Penicillin/streptomycin | Gibco | 15070-063 | |
| Alginate lyase | Sigma Aldrich | A1603 | |
| A1603 | Sigma Aldrich | C9697 | From Clostridium histolyticum |
| Bovine serum albumin | MP Biomedicals | 103700 | |
| Fetal bovine serum | Gibco | Gibco | |
| ITS solution | Roche | 11074547001 | Insulin/transferrin/sodium selenite |
| Cytokeratin 8 antibody | Developmental Studies Hybridoma Bank | TROMA-1 | Use at 1:200 |
| DAPI | Vector Labs | H1200 | |
| Bromodeoxyuridine | Sigma | B50002-1G | Final concentration 10 μM |
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