A Simple Method for Classification of Cell Death by Use of Thin Layer Collagen Gel for the Detection of Apoptosis and/or Necrosis after Cancer Chemotherapy

Abstract

To assess the efficacy of cancer chemotherapy, an important index is apoptosis of the target cells, which can usually be confirmed by electron microscopy (EM). We established a new experimental technique, whereby cancer cells (MKN45) were distributed in thin collagen gel as one or two cell layers, and cultured with anti-cancer drugs (5-FU and CDDP). The cells were stained with fluorescent Hoechst 33258 (Ho) and photographed, then with hematoxylin and eosin (H&E) and again photographed, and processed for EM. This approach allowed us to characterize the patterns of death of single cells in detail. There were six patterns of cell damage: two patterns of apoptosis, early peripheral condensation of chromatin and late apoptotic bodies, two patterns of necrosis, cytoplasmic swelling and washed-out images, and two further patterns, with morphological features of both apoptosis and necrosis, neither classified into necrosis nor apoptosis. The results show that cell death patterns can be mostly determined by combining observations of Ho and H&E-stained cells without the necessity for EM observation.

Transfection efficiency and toxicity of polyethylenimine in differentiated Calu-3 and nondifferentiated COS-1 cell cultures

“…An important cellular event during apoptosis is the cleaving of chromosomal DNA and packing in compartments, preparing the cell for breakdown into apoptotic bodies that can be efficiently cleared by adjacent cells without further damaging the tissue or triggering an immune response. 27,28 When the cells are fixed with formaldehyde and the nuclei are stained with a fluorescent dye, the formation of apoptotic nuclei can be visualized by fluorescence microscopy. An often-used nuclear dye is Hoechst 33258, and incubation with DMSO (5% or more) or serum starvation is used as a positive control for apoptosis induction in cells.…”

Evaluation of Carcinoembryonic Antigen mRNA in Living, Necrotic, and Apoptotic Gastric Cancer Cells by Reverse Transcriptase?Polymerase Chain Reaction

“…In terms of a cancer cell death condition such as apoptosis, MKN45 induced by culturing with the antitumor cytokines, TNFα ϩ IFNγ 13 or the anticancer drugs, 5FU ϩ CDDP, 12 showed both positive CEA and GAPDH mRNA expression, but there were some differences. The mRNA expression of MKN45 cultured with 5FU ϩ CDDP showed a tendency to decrease, but that cultured with TNFα ϩ IFNγ did not.…”

“…For apoptotic cell (AP) death, after preincubation with culture medium at 37°C for 24 h, MKN45 (1 ϫ 10 6 ) was recultured with 200 µg of 5-fluorouracil (5FU) and 40 µg of cisplatin (CDDP) in 2 ml medium (5FU 100µg/ ml ϩ CDDP 20 µg/ml) for 24 or 48 h, 12 and defined as the FC 24 h and FC 48 h groups, respectively. MKN45 was also recultured with 100 ng/ml of tumor necrosis factor (TNF)α and 100 ng/ml of interferon (IFN)γ for 24 or 48 h, 13 and defined as the TI 24 h and TI 48 h groups, respectively.…”

Leucovorin and fluorouracil plus oxaliplatin or leucovorin and fluorouracil plus irinotecan as individualized first-line therapy based on a drug sensitivity test

“…In planning individualized chemotherapy, the drug sensitivity of the tumor cells is a key issue in assessing the anti-tumor effect of the anticancer drugs to be used. The CD-DST is a method of evaluating drug sensitivity using isolated, 3-dimensionally cultured tumor cells in a small collagen gel droplet (12). This method offers the following advantages: i) a high success rate in testing due to the micro-3-dimensional culture; ii) the ability to work with a small quantity of specimen; iii) the ability to evaluate the anti-tumor effect of drugs in clinically equivalent doses; and iv) the ability to accurately evaluate anti-cancer effects using an image analysis device when fibroblast contamination is less than 67% (4).…”