The present study includes proof concept of morphology-based cell separation and development of automatic cell separation system. 3D culture environment with a specific extracellular matrix regulates cellular function and phenotype. In addition, cancer cell morphology changes depending on its malignancy in 3D culture environment. The cell separation system in 3D culture environment should need to obtain the cells according to its morphology, which includes cell phenotypes. Recently, we developed gelatin-based photodegradable hydrogels, and applied this hydrogels to optical cell separation. The target cells in the photodegradable hydrogels were successfully separated by the optical cell separation, the separated cells was growth on another dish. On the other hand, we recently developed the predication model of stem cell differentiation by image analysis. The image analysis technique and the photodegradable gelatin hydrogels are included in the automated morphology-based cell separation system in the 3D culture environment.

For forming cell encapsulated-photodegradable hydrogels, suspension of cells including heterogeneous population is mixed with pregel solutions and cells are encapsulated in the gelatin-based photodegradable hydrogels. After the culture in 3D environment, microscopic images of the cells are captured. The captured images are analyzed to distinguish the target cells from the other cells by using the image analysis algorithm, which we previously developed for analyzing stem cells. The hydrogels around the target area is irradiated with light (365nm). The cells in the irradiated area are collected by automated pipetting system. We developed automated system for this optical cell separation procedure, including cultivation, image acquisition, image analysis, light irradiation, and pipetting for cell collection.

We demonstrated automated optical cell separation using the model culture system. Normal gastric mucosal cells were cultured in the photodegradable hydrogels. After cultivation for 1 week, the cells were irradiated the light for 5 to 20 min. The cells in the irradiated area were collected by automated pipetting and transferred into a collection dish. The collected cells were viable and attached in the collection dish after collection.

We are currently developing an automated image analysis algorithm to distinguish cancer cells from normal cells under 3D environment. The automated optical cell separation system with image analysis algorithm will be applied to the establishment of novel cancer-cell lines from clinical samples such as biopsy tissue.

Citation Format: Hirofumi Matsui, Shinji Sugiura, Masato Tamura, Toshiyuki Kanamori, Toshiyuki Takagi, Taku Satou, Ryuji Kato, Kei Kanie, Mayu Shibuta. Development of cellular morphology-based separation system for three-dimensional culture [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5788. doi:10.1158/1538-7445.AM2017-5788