Growth characterization of neo porcine cartilage pellets and their use in an interactive culture model.
OBJECTIVE
The aim of this study was to evaluate the growth characteristics of freshly isolated porcine chondrocytes in high-density pellet cultures and to preliminary investigate their use in an interactive in vitro model with synovial fibroblast cell lines to study rheumatoid arthritis (RA).


METHODS
1.8x10(6) chondrocytes/cm2 were seeded in 48-multiwell plates. Thickness, cell number and cell distribution in pellet cross sections were documented over a 22-day-long period. Alcian blue staining, type I and type II collagen staining, real-time reverse transcriptase polymerase polymerase chain reaction (RT-PCR) and high performance liquid chromatography (HPLC) were used to characterize cartilage extracellular matrix (ECM) formation, and cell proliferation was demonstrated by Ki67 staining. Furthermore, 2-week-old chondrocyte pellets were co-cultured for additional 2 weeks with two human synovial fibroblast cell lines derived from a normal donor (non-invasive cell line) and a RA patient (invasive-aggressive (IA) cell line), respectively.


RESULTS
Chondrocyte pellets from 11 individual preparations showed a significant increase in pellet thickness from 44+/-19 microm (day 3) to 282+/-19 microm (day 22). Calculation of chondrocyte distribution, cell number and pellet thickness indicated that pellet growth was due to ECM formation and not cell proliferation. This was also confirmed by low numbers of Ki67 positive chondrocytes and absence of cell clusters. HPLC, messenger RNA-analysis, histochemistry and antibody staining verified the expression of ECM components such as type II collagen, whereas type I collagen expression was very low. In contrast to the non-aggressive synovial fibroblast cell line, the IA synovial fibroblast cell line clearly showed cartilage invasion.


CONCLUSIONS
Pellet formation of freshly isolated chondrocytes followed a reproducible developmental kinetics and showed typical immature hyaline cartilage properties. Such uniform cartilage pellets are very useful as a substrate for interactive cell culture models that simulate diseases like RA.
Lübke
Carsten
C
VasoTissue Technologies GmbH, Department of Research and Development, Tucholskystrasse 2, 10117 Berlin, Germany. carsten.luebke@vasotissue.com
Ringe
Jochen
J
Krenn
Veit
V
Fernahl
Gabriele
G
Pelz
Stine
S
Kreusch-Brinker
Rüdiger
R
Sittinger
Michael
M
Paulitschke
Manrico
M
D000818:Animals; D001172:Arthritis, Rheumatoid / Q000517:prevention & control; D002358:Cartilage, Articular / Q000166:cytology*; D002478:Cells, Cultured; D019902:Chondrocytes / Q000166:cytology / Q000378:metabolism*; D018920:Coculture Techniques; D024043:Collagen Type II / Q000096:biosynthesis; D005109:Extracellular Matrix / Q000378:metabolism*; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism*; D007150:Immunohistochemistry; D008954:Models, Biological; D016133:Polymerase Chain Reaction; D013552:Swine; D023822:Tissue Engineering / Q000379:methods*
D024043:Collagen Type II