title: The challenge of culturing human colorectal tumor cells: establishment of a cell culture model by the comparison of different methodological approaches.
abstract: BACKGROUND
  Because colorectal cancer is a significant cause of morbidity and mortality in the Western population, knowledge of the molecular and biological alterations associated with its development is important. Since primary human colon cancer cultures from fresh tumor tissue are technically difficult to obtain, experiments in most laboratories are performed on colon epithelial cell lines, but these represent just one stage of tumor progression. Only primary cultures of neoplastic colonocytes may reflect the actual responsiveness of tumors at certain developmental stages to antitumor agents.
  
  
  METHODS
  This paper analyzes several critical points concerning primary cultures, ranging from cell isolation to culture conditions, and compares different methodological approaches to isolate and cultivate a pure fraction of viable tumor cells. Samples of resected colorectal cancers were collected from 20 patients (stage T3 or T4). We compared in vitro several approaches of tissue disaggregation including mechanical disaggregation and enzymatic dissociation with trypsin or collagenase. Isolated cells were maintained in a short-term serum-free culture system. Evaluation of the purity and tumoral nature of isolated cells was performed by immunochemistry.
  
  
  RESULTS
  We established the antibiotic concentration necessary during transport and washing of the specimens to prevent microbial overgrowth. We demonstrated that the number of viable cells was dependent on the dissociation method used. Mechanical disaggregation is not a valid dissociation method because of the high mortality of cells and might be used only in samples for molecular analysis. Comparison of the enzymatic digestion procedures showed that digestion with trypsin allowed the highest recovery of viable cells.
  
  
  CONCLUSIONS
  In this paper we analyzed several critical aspects of cell culture procedures and designed a methodological approach suitable for functional studies of colorectal cancer.
authors.affiliation: Department of Medicina della Procreazione e dell'Età Evolutiva, Laboratory of Immunology, University of Pisa, Pisa, Italy.
authors.forename: Alessandra
authors.initials: A
authors.lastname: Failli
authors.forename: Rita
authors.initials: R
authors.lastname: Consolini
authors.forename: Annalisa
authors.initials: A
authors.lastname: Legitimo
authors.forename: Roberto
authors.initials: R
authors.lastname: Spisni
authors.forename: Maura
authors.initials: M
authors.lastname: Castagna
authors.forename: Antonella
authors.initials: A
authors.lastname: Romanini
authors.forename: Gaetano
authors.initials: G
authors.lastname: Crimaldi
authors.forename: Paolo
authors.initials: P
authors.lastname: Miccoli
mesh_terms: D000368:Aged; D000369:Aged, 80 and over; D018929:Cell Culture Techniques / Q000379:methods*; D045744:Cell Line, Tumor; D002469:Cell Separation / Q000379:methods; D002470:Cell Survival; D015179:Colorectal Neoplasms / Q000473:pathology; D003470:Culture Media; D005260:Female; D006801:Humans; D007150:Immunohistochemistry; D008297:Male; D008875:Middle Aged
chemical_list: D003470:Culture Media