Primary murine small intestinal epithelial cells, maintained in long-term culture, are susceptible to rotavirus infection.
We describe a method for long-term culture of primary small intestinal epithelial cells (IEC) from suckling mice. IEC were digested from intestinal fragments as small intact units of epithelium (organoids) by using collagenase and dispase. IEC proliferated from organoids on a basement-membrane-coated culture surface and remained viable for 3 weeks. Cultured IEC had the morphologic and functional characteristics of immature enterocytes, notably sustained expression of cytokeratin and alkaline phosphatase. Few mesenchymal cells were present in the IEC cultures. IEC were also cultured from adult BALB/c mice and expressed major histocompatibility complex (MHC) class II antigens for at least 48 h in vitro. Primary IEC supported the growth of rhesus rotavirus (RRV) to a greater extent than a murine small intestinal cell line, m-IC(cl2). Cell-culture-adapted murine rotavirus strain EDIM infected primary IEC and m-IC(cl2) cells to a lesser extent than RRV. Wild-type EDIM did not infect either cell type. Long-term culture of primary murine small intestinal epithelial cells provides a method to study (i) virus-cell interactions, (ii) the capacity of IEC to act as antigen-presenting cells using a wide variety of MHC haplotypes, and (iii) IEC biology.
Macartney
K K
KK
Section of Infectious Diseases, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania 19104, USA. macartney@email.chop.edu
Baumgart
D C
DC
Carding
S R
SR
Brubaker
J O
JO
Offit
P A
PA
D000222:Adaptation, Physiological; D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D001485:Basement Membrane / Q000378:metabolism; D015415:Biomarkers / Q000032:analysis; D018929:Cell Culture Techniques / Q000379:methods; D002455:Cell Division; D002460:Cell Line; D002469:Cell Separation; D048429:Cell Size; D002470:Cell Survival; D002478:Cells, Cultured; D020895:Enterocytes / Q000166:cytology / Q000201:enzymology / Q000378:metabolism / Q000821:virology*; D005260:Female; D000949:Histocompatibility Antigens Class II / Q000378:metabolism; D007421:Intestine, Small / Q000166:cytology / Q000201:enzymology / Q000378:metabolism / Q000821:virology*; D007633:Keratins / Q000032:analysis; D008297:Male; D008648:Mesoderm / Q000166:cytology; D051379:Mice; D008807:Mice, Inbred BALB C; D012401:Rotavirus / Q000502:physiology*; D013997:Time Factors
D015415:Biomarkers; D000949:Histocompatibility Antigens Class II; D007633:Keratins; D000469:Alkaline Phosphatase