In vitro mammary gland model: establishment and characterization of a caprine mammary epithelial cell line | In vitro model mlečne žleze: vzpostavitev in določitev značilnosti epitelne celične linije iz kozje mlečne žleze

Inglés. Demanding transcriptomic studies in combination with challenging experiments in livestock animal species could be replaced by good in vitro models mimicking the function of ruminant mammary gland. The objective of our study was to establish epithelial cell line obtained from primary cell culture of lactating goat mammary gland. Mammary tissue from lactating Saanen goat (Capra hircus) was digested in collagenase and hyaluronidase solution and plated on plastic flasks. When growing on plastic, typical cobblestone morphology of epithelial cells and larger irregularly shaped cells, corresponding to myoepithelial cells were observed. When growth medium was supplemented with lactogenic hormones (insulin, hydrocortisone, and prolactin) and cells were cultured on plastic for extended period of time at high density, dome-like structures appeared as a result of cell to cell contact induced differentiation. Immunofluorescence staining using antibodies against smooth muscle α-actin, vimentin and various cytokeratins were used to distinguish between different cell types. Cell types of epithelial and myoepithelial cells were confirmed. Complete differentiation of cells was achieved when growing them on a commercial basal membrane matrix preparation which contains laminin, collagen IV, and various growth factors. Cells grown on basal membrane matrix in growth medium supplemented with lactogenic hormones differentiated morphologically and functionally. Spherical structures that resembled the alveoli of lactating mammary gland were observed. Reverse transcription PCR (RT-PCR) was performed on the total RNA extracted from the cultured cells in order to detect the potentially present milk protein mRNAs.

Esloveno. Zahtevne transkriptomske študije, ki vključujejo poskuse na živih živalih, je mogoče nadomestiti z ustreznimi in vitro modeli. Cilj naše raziskave je bil vzpostaviti celično linijo epitelnih celic, pridobljenih iz primarne celične kulture kozje mlečne žleze v laktaciji. Žlezno tkivo koze (Capra hircus) sanske pasme smo razgradili v raztopini kolagenaze in hialuronidaze in nacepili v plastične posodice. Pri rasti na plastični podlagi so se pojavile značilne epitelne strukture v obliki tlakovcev in večje celice nepravilnih oblik, ki so po zunanjosti ustrezale mioepitelnim celicam. Ob dodatku laktogenih hormonov (inzulin, hidrokortizon, prolaktin) v medij in po daljšem obdobju rasti ter ob visoki gostoti celic na plastični podlagi so se oblikovale kupolaste strukture, ki so posledica diferenciacije zaradi medceličnih interakcij. Za karakterizacijo različnih celičnih tipov smo uporabili imunofluorescenčno barvanje za α-aktin gladkih mišic, vimentin in različne citokeratine. Z barvanjem smo potrdili prisotnost epitelnih in mioepitelnih celic. Popolno diferenciacijo celic smo dosegli z gojenjem na komercialno pripravljenem matriksu, ki posnema bazalno membrano in vsebuje laminin, kolagen IV in različne rastne faktorje. Celice so se na podlagi iz ekstracelularnega matriksa in ob dodatku laktogenih hormonov morfološko in funkcionalno diferencirale. Nastale so sferične strukture, podobne alveolam mlečne žleze v laktaciji. Z reverzno verižno reakcijo s polimerazo (RT-PCR) smo na izolirani RNA preverili prisotnost mRNA za mlečne proteine.