title: The liquid overlay technique is the key to formation of co-culture spheroids consisting of primary osteoblasts, fibroblasts and endothelial cells.
abstract: OBJECTIVE
  The 3-dimensional (3-D) culture of various cell types reflects the in vivo situation more precisely than 2-dimensional (2-D) cell culture techniques. Spheroids as 3-D cell constructs have been used in tumor research for a long time. They have also been used to study angiogenic mechanisms, which are essential for the success of many tissue-engineering approaches. Several methods of forming spheroids are known, but there is a lack of systematic studies evaluating the performance of these techniques.
  
  
  METHODS
  We evaluated the performance of the hanging drop technique, carboxymethyl cellulose technique and liquid overlay technique to form both mono- and co-culture spheroids consisting of primary osteoblasts, fibroblasts and endothelial cells. The performance of the three techniques was evaluated in terms of rate of yield and reproducibility. The size of the generated spheroids was determined systematically.
  
  
  RESULTS
  The liquid overlay technique was the most suitable for generating spheroids reproducibly. The rate of yield for this technique was between 60% and 100% for monoculture spheroids and 100% for co-culture spheroids. The size of the spheroids could be adjusted easily and precisely by varying the number of seeded cells organized in one spheroid. The formation of co-culture spheroids consisting of three different cell types was possible.
  
  
  CONCLUSIONS
  Our results show that the most suitable technique for forming spheroids can vary from the chosen cell type, especially if primary cells are used. Co-culture spheroids consisting of three different cell types will be used to study angiogenic phenomena in further studies.
authors: Department of Trauma, Hand and Reconstructive Surgery, Saarland University, Homburg, Germany. johann-wolfgang.metzger@uks.eu
authors: Wolfgang
authors: W
authors: Metzger
authors: Daniela
authors: D
authors: Sossong
authors: Annick
authors: A
authors: Bächle
authors: Norbert
authors: N
authors: Pütz
authors: Gunther
authors: G
authors: Wennemuth
authors: Tim
authors: T
authors: Pohlemann
authors: Martin
authors: M
authors: Oberringer