**Is Anticancer Vaccine Possible: Experimental Application of Produced mRNA Transfected Dendritic Cells Derived from Enriched CD34+ Blood Progenitor Cells**

Paula Lazarova, Gunnar Kvalheim and Krassimir Metodiev

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/51544

#### **1. Introduction**

76 Immunodeficiency

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The dendritic cells (DCs) are the most powerful antigen-presenting cells (APC) specialized to induce and regulate immune responses (1,2). The clinical use as cellular adjuvants in vaccination strategies has been aided by the development of methodologies to generate large production of these cells in culture. DCs can be grown ex-vivo from blood monocytes (3,4,5) or enriched CD34+ progenitors (6,7), using combinations of several cytokines/growth factors. Since our laboratory in Oslo routinely uses enriched CD34+ stem cells as stem cell support following high dose radio- and chemotherapy, it was of interest to test if such cells also could be applied for vaccine purposes (8,9), with a long term strategy of combining the two forms of therapy.

There are some publications indicating that CD34+ derived DC may work more efficiently as APC than those derived from monocytes (10), and recent data confirm that vaccine programs using CD34+ cell derived DCs lead to improved clinical results (11).

However, most in vitro culture systems for production of DCs include serum (9,11,12).

Since DCs are able to take up and process serum–derived antigens that are present in the cell cultures, such DC can when injected create unwanted reactions in the patients, in particular when fetal calf serum (FCS) is used. Thus, serum-free culturing condition is preferable, but in most previous culture experiments these conditions resulted in a lower yield of DCs (13,14).

Recently we reported a protocol for producing DCs from monocytes by use of gaspermeable Teflon bags and serum-free medium (15). We have used in the present study this

experience and have developed a similar serum-free culture system for CD34+ cell derived DC and investigated the optimal immunological properties of these cells.

Is Anticancer Vaccine Possible: Experimental Application of Produced mRNA Transfected Dendritic Cells Derived from Enriched CD34+ Blood Progenitor Cells 79

(CellGenix, Freiburg, Germany) with serum containing DMEM/10% FCS medium, serumfree CellGro/SCGM medium or CellGro/SCGM/25% human albumin respectively. The following cytokine cocktail was added: GM-CSF 1000u/ml, IL-4 500u/ml, TNF-α 50ng/ml, Flt-3L 150ng/ml and SCF 50ng/ml. The bags were cultured for 14 days at 37ºC/5% CO2. To keep a cell concentration of 105cells per ml through the entire culture period, re-feeding of the cells with culture medium employing equal concentration of cytokines was performed at weekly intervals. The serum-free medium cultures were from day 7, supplemented with

**Figure 2.** Expansion fold on Day 14. Different initial concentrations of CD34+ cells incubated in

**2.4. Isolation of mRNA from cell line of human prostate cancer origin** 

different culture medium. Cell concentration 105/ml are expanded better than 104/ml especially in serum

Prostate cancer cell line DU 145 obtained from American Type Culture Collection (ATCC), was cultured in RPMI 1640 supplemented with 10% FCS. The method employed for isolation of mRNA from the tumor cell line has been described earlier (15). Briefly, 5x107 cells were washed with cold PBS and transferred to a 1.5 ml microtube. Five hundred microliter ice-cold 2% IGEPAL (polyoxyethylene 9 nonylphenyl ether) (Sigma-Aldrich) was added to lyse the cells. The supernatant containing the cytosol fraction was obtained after centrifugation (10000xG for 1 minute at 4°C) and transferred to a 1.5 ml tube placed on a cooling block (4°C). To the supernatant 80 μl 10% LiDS (Lithium Lauryl Sulfate) (Sigma-Aldrich), 80 ul 5M LiCl (Lithium Chloride) (Sigma-Aldrich) and 0.5 ml Lysis & Binding Solution (Geno Vision) was added. Samples were frozen and stored at –80ºC until use. Isolation and purification of mRNA from the frozen or fresh samples were prepared in a GenoMTM-48 Robotic Workstation (Genovision AS, Oslo, Norway) following the procedure

CellGro DC medium instead of CellGro SCGM medium.

free medium
