title: Effective recovery of highly purified CD326(+) tumor cells from lavage fluid of patients treated with a novel cell-free and concentrated ascites reinfusion therapy (KM-CART).
abstract: For the production of tumor-specific vaccines, including dendritic cell (DC) vaccines, the tumor cells themselves are an ideal source. Floating tumor cells in the ascites fluid from patients with malignant ascites are a good candidate source, but it is not easy to obtain pure tumor cells from ascites because of various types of cell contamination as well as protein aggregates. We here report an effective method to recover pure tumor cells from malignant ascites. We used lavage fluid from 13 patients with malignant ascites who were treated with modified cell-free and concentrated ascites reinfusion therapy (KM-CART). Cellular components were separated from the lavage fluid by centrifugation, enzymatic digestion and hemolysis. Tumor cells were purified by depleting CD45(+) leukocytes with antibody-conjugated magnetic beads. The tumor cell lysate was extracted by freeze-and-thaw cycles. The mean obtained total cell number was 7.50 × 10(7) cells (range 4.40 × 10(6)-2.48 × 10(8) cells). From this fraction, 6.39 × 10(6) (range 3.23 × 10(5)-2.53 × 10(7)) CD45(-) cells were collected, and the tumor cell purity was over 80 % defined as CD45(-)CD326(+). A sufficient amount of tumor lysate, average  = 2416 μg (range 25-8743 μg), was extracted from CD45(-)CD326(+) tumor cells. We here established an effective method to produce highly purified tumor cells from KM-CART lavage fluid. The clinical feasibility of this simple preparation method for generating tumor lysate should be examined in clinical studies of DC vaccines.
authors.affiliation: R&D Laboratory for Innovative Biotherapeutics, Graduate School of Pharmaceutical Sciences, Kyushu University, 6 floor, Collaborative Research Station I, 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812-8582 Japan ; Tella, Inc., Tokyo, Japan.
authors.forename: Yukino
authors.initials: Y
authors.lastname: Kimura
authors.affiliation: R&D Laboratory for Innovative Biotherapeutics, Graduate School of Pharmaceutical Sciences, Kyushu University, 6 floor, Collaborative Research Station I, 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812-8582 Japan.
authors.forename: Yui
authors.initials: Y
authors.lastname: Harada
authors.affiliation: R&D Laboratory for Innovative Biotherapeutics, Graduate School of Pharmaceutical Sciences, Kyushu University, 6 floor, Collaborative Research Station I, 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812-8582 Japan.
authors.forename: Noriko
authors.initials: N
authors.lastname: Yasuda
authors.affiliation: AllGene, Inc., Kanagawa, Japan.
authors.forename: Takefumi
authors.initials: T
authors.lastname: Ishidao
authors.affiliation: Tella, Inc., Tokyo, Japan.
authors.forename: Seiichi
authors.initials: S
authors.lastname: Yusa
authors.affiliation: Ascites Treatment Center, Kanamecho Hospital, Tokyo, Japan.
authors.forename: Keisuke
authors.initials: K
authors.lastname: Matsusaki
authors.affiliation: R&D Laboratory for Innovative Biotherapeutics, Graduate School of Pharmaceutical Sciences, Kyushu University, 6 floor, Collaborative Research Station I, 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812-8582 Japan.
authors.forename: Yoshikazu
authors.initials: Y
authors.lastname: Yonemitsu
keywords: Ascites; CART; Tumor lysate