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Kimura Y, Harada Y, Yasuda N, et al. 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). Springerplus. 2015;4:780doi: 10.1186/s40064-015-1508-3.
Kimura, Y., Harada, Y., Yasuda, N., Ishidao, T., Yusa, S., Matsusaki, K., & Yonemitsu, Y. (2015). 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). SpringerPlus, 4780. https://doi.org/10.1186/s40064-015-1508-3
Kimura, Yukino, et al. "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)." SpringerPlus vol. 4 (2015): 780. doi: https://doi.org/10.1186/s40064-015-1508-3
Kimura Y, Harada Y, Yasuda N, Ishidao T, Yusa S, Matsusaki K, Yonemitsu Y. 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). Springerplus. 2015 Dec 17;4:780. doi: 10.1186/s40064-015-1508-3. eCollection 2015. PMID: 26702369; PMCID: PMC4683161.
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Springerplus. 2015 Dec 17;4:780. doi: 10.1186/s40064-015-1508-3. eCollection 2015.

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).

SpringerPlus

Yukino Kimura, Yui Harada, Noriko Yasuda, Takefumi Ishidao, Seiichi Yusa, Keisuke Matsusaki, Yoshikazu Yonemitsu

Affiliations

  1. 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.
  2. 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.
  3. AllGene, Inc., Kanagawa, Japan.
  4. Tella, Inc., Tokyo, Japan.
  5. Ascites Treatment Center, Kanamecho Hospital, Tokyo, Japan.

PMID: 26702369 PMCID: PMC4683161 DOI: 10.1186/s40064-015-1508-3

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.

Keywords: Ascites; CART; Tumor lysate

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