**Author details**

86 Immunodeficiency

The use of gas permeable bags for ex-vivo production has several advantages when compared to production in culture flasks. The bag system is closed and reduces the risk of contamination. DCs produced in Teflon bags do not attach to the surface and can easily be concentrated by centrifugation without any extra steps. It also facilitates large-scale production, which can be divided into aliquots containing cells with identical properties. We have shown that DCs can efficiently be produced in suspension using gas permeable Teflon bags. When CD34+ progenitors are cultured in flasks, usually the cell concentration is 104/ml. In our system we have shown that optimal cell concentration is 105/ml, which give a

DCs have been loaded with several antigens, such as tumor lysate, peptides, proteins, DNA and mRNA. As a source of antigens, the major limitation of using lysate, proteins or peptides isolated from patients' tumor cells is the amount of tumor tissue or the purity of the tumor specimens. The use of nucleic acids, either DNA or RNA, would overcome this practical limitation. As mRNA is a safer alternative due to its limited ability to cause permanent genetic alterations in the host, it appears to be more attractive to be used than DNA transfection (23). For this purpose, a vector-free transfection system based on square-wave electroporation to transfer mRNA into DCs has been developed (17). This method is currently successfully used in the clinic to produce mRNA-transfected monocyte-derived DCs (15). We here demonstrate that this method also resulted in efficient transfection of mRNA into immature CD34+ cell derived DCs without significantly affecting the survival of the cells.

Generally the antigen-stimulating capacity of DCs has been evaluated employing alloreactive T cells or responses against recall antigens. We have used priming of autologous T cell against antigens encoded by a prostate tumor cell line in order to evaluate the immunostimulatory role of the transfected DCs. The ELISPOT assay was used to detect and quantify of single T lymphocyte forming cytokine spots after antigen contact in vitro. The ELISPOT assay is a more stringent system for testing both the efficacy of transfection and the processing and antigen-stimulating capacity of transfected DCs. Our results show that CD34+ cells derived DCs, grown in serum free conditions in clinical scale productions reproducibly are capable of inducing a tumor specific immune response. These results are

The finding and results from the present study allows us to proceed with a clinical protocol for application of CD34+ derived DCs for cancer vaccine. Possible attractive candidates for such an approach are relapsed Hodgkin's patients (16) and other patients that have previously been

Experiment Cell Gro/SCGM&25% HA DMEM / 10% FCS Cell Gro / SCGM / DC

1 1.6 / 61% 9.0 / 97% 8.6 / 94% 2 1.2 / 67% 7.8 / 98% 7.1 / 93% 3 1.8 / 73% 8.8 / 97% 7.1 / 84%

fold / viability fold / viability fold / viability

similar to what was seen in our previous study using monocyte-derived DCs

treated with auto transplantation and with spared frozen samples of CD34+ cells.

**Table 1.** Expansion fold and viability on Day 14

10-fold reduction in the amount of medium and cytokines used.

Paula Lazarova1,2,\*, Gunnar Kvalheim1, Krassimir Metodiev3 *1Dept. Cellular Therapy, University Rikshospital-Radiumhospital, Oslo, Norway, 2Clinical laboratory, District Hospital "St. Anna", Varna, Bulgaria, 3Dept. Preclinical & Clinical Sciences, Medical University, Varna, Bulgaria* 
