Dextran sulphate crowding and sodium deoxycholate lysis of primary breast fibroblast cells achieve extracellular matrix deposition and decellularization for breast cancer stem cell culture

Abstrak Latar belakang: Lingkungan mikro yaitu sel stromal dam matriks ekstraseluler saat ini dinyatakansebagai kontributor dalam perkembangan tumor. Beberapa penelitian telah mengembangkan matriksekstraseluler yang mendukung perkembangan sel in vitro. Matriks ekstraseluler adalah suatu komplekssusunan supramolekuler dari berbagai macam glycoprotein dan proteoglycan. Matriks ekstraselulermenyediakan integritas jaringan, bertindak sebagai scaffold alami tempat sel melekat dan berinteraksiserta berperan sebagai reservoir pertumbuhan sel. Penelitian ini bertujuan untuk mendapatkan deposisidan deselularisasi yang optimal pada matriks ekstraseluler. Metode: Dalam penelitian ini, kami mengembangkan cells crowder untuk meningkatkan deposit matriksekstraseluler dari kultur sel primer fibroblast payudara yang diperoleh dari spesimen hasil operasimammoplasty. Dextran 500 kDa ditambahkan dalam media kultur DMEM lengkap yang telah ditambahkan0.5% FBS dan 100μM L-ascorbic acid 2-phosphate. Setelah tujuh hari, sel dilisis dengan menggunakanSodium Deoxycolate (DOC). Hasil: Deposisi matriks ekstraseluler dan proses deselulerisasi dari sel primer fibroblas payudara dapatterdeteksi dengan menggunakan antibodi Rabbit anti human fibronectin yang selanjutnya ditambahkandengan anti rabbit IgG yang telah dikonjugasi dengan Alexa Fluor 488. Kesimpulan: Penambahan dextran sulfat dan prosesing lysis dengan sodium deoxycolate dapatmeningkatkan deposisi dan menghasilkan deselularisasi matriks ekstraseluler. (Health Science Journalof Indonesia 2015;6:43-7) Kata kunci: matriks ekstra selular, kanker mammae, stem cell, sel fibroblast Abstract Background: The microenvironment including stromal cells and extracellular matrix (ECM) is now consideredan active contributor to tumor progression. Certain studies have developed ECM which supports a suitable cellulargrowth in vitro. The ECM is a complex supramolecular assembly of a variety of glycoproteins and proteoglycans.Extracellular matrix provides tissue integrity, acts as a native scaffold for cell attachment and interaction and alsoserves as reservoir for growth factors. The aim of this experiment w asto achieve the deposition and decellularizationof ECM. Methods: Cells crowder have been developed to increase ECM deposit in the primary breast fibroblast cells layerobtained from isolation of single cell from breast mammoplasty specimen. Five hundred kDa dextran was addedinto DMEM medium containing 0.5% fetal bovine serum (FBS) and 100 μm of L-ascorbic acid 2-phosphate. Afterseven days, cells were lysed by sodium deoxycholate (DOC). Results were observed in a fluorescence microscope. Results: Extracellular matrix deposition and decellularization of primary breast fibroblast cells were detected byusing extracellular matrix protein, fibronectin via rabbit anti human fibronectin and anti-rabbit IgG conjugated withAlexa Fluor 488. Conclusion: Dextran sulphate increased extracellular matrix deposit in primary breast fibroblast cell layer and thetreatment with sodium deoxycolate lysed cells resulted in extracellular matrix decellularization. (Health ScienceJournal of Indonesia 2015;6:43-7) Keywords: extracellular matrix, breast cancer stem cell, breast fibroblast cell