Transplantation of Genetically Engineered Cardiac Fibroblasts Producing Recombinant Human Erythropoietin to Repair the Infarcted Myocardium - Genetic Engineering: Recent Developments in Applications - page 190

supernants were first incubated at room temperature with a Superfect cationic
transfection reagent (QIAGEN, Westburg Ltd., Be'er-Sheva, Israel) to a final con-
centration of 10 µ g/ml for 15 minutes [17]. Finally, the viral stock was added to
70-80% confluent BALB/3T3 or cardiac fibroblast culture. After 24 h incuba-
tion at 37°C, puromycin selection was applied (2.5 µ g/ml). Transduction effi-
ciency was approximately 80%, as evaluated qualitatively by relative confluence
determination (before and after puromycin selection). Twenty-four hour culture
supernants were collected for EPO assessment. Supernants were stored at -20°C
for future experiments.
Assessment of ePo Production and Activity
The presence of rhEPO in transduced culture supernants was analyzed by a com-
mercial ELISA kit (R&D Systems, Minneapolis, MN, USA). EPO concentration
was normalized to 106 cells for 24 h.
EPO biological activity was analyzed using the EPO-sensitive human erythro-
leukemia cell line TF-1, in which proliferation and metabolic activity is EPO-
dependent [18].
TF-1 cells were seeded in 96-well plates (1 × 105 cell per well in 100 µl of
RPMI medium). A total of 100 µl of previously collected culture supernants,
controls or standards (0.01 to 5 IU/ml rhEPO; Eprex, Beerse, Belgium) were
added to each well. On day 5 of the experiment, TF-1 relative cell number was
analyzed using colorimetric XTT assay kit (Biological Industries, Beth Haemek,
Israel). The XTT assay is based on the ability of metabolically active (live) cells to
reduce tetrazolium salt (XTT) to orange-colored compounds of formazan. EPO
activity in supernants from rhEPO-transduced fibroblast culture was calculated
from XTT rhEPO standard calibration curve. EPO relative biological activity was
determined by EPO activity level/EPO protein level (measured by ELISA) ratio.
This amount-normalized protein ratio was used for comparison of biological ac-
tivity between expressed EPO (produced from transduced cells) and recombinant
protein.
oxidative stress
After 6 days in culture, isolated rat neonatal cardiomyocytes were pretreated with
supernants collected from rhEPO-transduced or non-transduced (for control)
cardiac fibroblasts for 24 h, after which H2O2 was directly added to culture me-
dium to a 150 µ M final concentration for 60 minutes, and the degree of apoptosis
was tested.