A unique in vitro model of xenogeneic heart transplantation using a micro-gravity based CO-culture system: heat shock protein-60 expression and apoptosis
Introduction A NASA derived microgravity based cell culture system could provide a unique in vitro model for studying xenogeneic heart transplantation. The microgravity environment maintains three-dimensional relationships important in tissue interactions. We applied this system to study apoptosis and expression of heat shock protein-60.
Materials and methods Brown Norway rat heart and spleen and pig spleen were harvested and sectioned into 1-2 mm pieces and loaded into bioreactors. Both syngeneic (rat heart and spleen) and xenogeneic (rat heart and pig spleen) models were created using multiple bioreactors. All bioreactors contained RPMI media and serum to maintain the tissue, and were incubated at 37°C for 10 days. Sections were stained using TUNNEL to identify apoptosis. Immunostains were also performed to identify pig splenocytes (CD45) and heat shock protein expression (HSP60). Digital image analysis was performed to quantify the stained area, nuclear number, interstitial space and histopathologic morphology. Western blotting was done to identify heat shock proteins in growth media.
Results Immunostaing for CD45 revealed attachment of pig splenocytes to the periphery of the rat cardiac tissue. Digital image analysis of the sections on which TUNNEL was performed displayed increasingly apoptosis under both experimental conditions with the passage of time. The syngeneic model however, showed significantly more apoptosis at each time point (P = 0.025). HSP60 was expressed in myocardial tissue decreasing from day 2 to day 9 (P = 0.00001) for syngeneic, and was increasing expressed over time in fibroblasts for the xenogeneic group P = 0.04. Western blots detected HSP60 in the media of both groups, but was significantly increased in the syngeneic group.
Conclusions The degree of apoptosis decreased dramatically with time in the syngeneic compared to the xenogeneic group. Histopathologic morphology showed striking differences in xenogeneic myocardial tissues due to intense fibroblast staining with HSP60. Fibroblasts are well documented to produce cytokines associated with HSP expression and apoptosis which play a major role in inflammation. This suggests that the bioreactor model may be useful for acute and chronic xenogeneic rejection studies.