Retaining cell–cell contact enables preparation and culture of spheroids composed of pure primary cancer cells from colorectal cancer

Abstract

Primary culture of the cancer cells from patients’ tumors can provide crucial information of individual tumors, yet the technology has not been optimized until now. We developed an innovative culture method for primary colorectal cancer cells, based on the principle that cell–cell contact of cancer cells was maintained throughout the process. When tumor tissue was dissociated into cell clusters, in which cell–cell contact was retained, they rapidly formed spheroids that we termed cancer tissue-originated spheroids (CTOSs). CTOSs of colorectal cancer consisted of highly purified and viable cancer cells, and they were prepared with high efficiency. In immunodeficient mice, CTOSs formed xenograft tumors that retained the features of the parental tumors. Moreover, CTOSs were able to be cultured and expanded in vitro using a 3D culture system and stem cell culture medium. This method allowed evaluation of chemosensitivity and signal pathway activation in cancer cells from individual patients. Easy preparation and culture of pure primary cancer cells provides an innovative platform for studying cancer biology and developing personalized medicine.

High-throughput screening with nanoimprinting 3D culture for efficient drug development by mimicking the tumor environment

“…Collagen gel was composed of cell-matrix type I-A (Nitta Gelatin, Osaka, Japan):10 Â DMEM (Sigma, Saint Louis, MO, USA):reconstitution buffer (50 mM NaOH, 260 mM NaHCO 3 , 200 mM HEPES) at a ratio of 8:1:1 (vol/vol/vol). In this study, we used two different CTOS spheroid lines (C45 and C132), which were obtained from two patients with colorectal cancer used in the previous study [24], expanded via transplantation into mice, and maintained with reported procedures [24] (Table S2). To seed cells evenly onto the NCPs, the obtained CTOS spheroids were mono-cellularized using the following procedures.…”

“…To construct the NCP drug screening system with cells derived from patient cancer tissues, patient-derived original cancer spheroids obtained by a recentlydeveloped CTOS method [24] were used. Briefly, colorectal cancer specimen samples were obtained from patients at the Osaka Medical Center for Cancer and Cardiovascular Diseases.…”

“…To achieve this, we employed the recently-developed Cancer TissueOriginated Spheroid (CTOS) method, which allows for highly efficient preparation of large amounts of primary spheroids with purified cancer cells, without contamination of host cells from patient cancer tissues or biopsy samples [24]. The CTOS method has numerous advantages; however, the CTOS spheroids are varied in shapes and size, causing difficulties in applying CTOS spheroids to the high-throughput drug screening directly.…”

Augmented Pentose Phosphate Pathway Plays Critical Roles in Colorectal Carcinomas

“…Undigested debris was removed by a 1,000- as well as a 500-μm nylon mesh, and finally cell clumps that did not pass through 100-μm nylon mesh were collected for primary culture [12]. The cell clumps formed cancer cell spheroids or otherwise became attached to the plastic dish on the following day with a success rate of almost 100% [12].…”

“…Undigested debris was removed by a 1,000- as well as a 500-μm nylon mesh, and finally cell clumps that did not pass through 100-μm nylon mesh were collected for primary culture [12]. The cell clumps formed cancer cell spheroids or otherwise became attached to the plastic dish on the following day with a success rate of almost 100% [12]. The culture medium was serum-free advanced DMEM/F-12 (GIBCO) supplemented with FGF-2 (10 ng/ml; ReproCell, Yokohama, Japan), ROCK inhibitor [13] (Y-27632, 10-20 μ M ; Wako Pure Chemical, Osaka, Japan), penicillin (100 mg/ml; Wako), streptomycin (0.1 mg/ml; Wako), gentamicin (50 μg/ml; Wako) and Fungizone (2.5 μg/ml; GIBCO).…”