Ultrastructural Study on Microtumor Spheroids from Human Malignant Glioma Specimens Maintained in Organotypic Culture

Shin, Hyung Jin; Chung, Chun Kee; Park, In Sung; Chung, Young Seob; Jung, Hee Won; Han, Dae Hee

Abstract

The Present study investigated not only the feasibility of organotypic spheroid culture system taken from human malignant gliomas, but also the similarities and differences between surgical specimens and cultured spheroids using light microscopy, electron microscopy, and flow cytometric examination. Surgically resected tumor specimens from eighteen human malignant gliomas were minced and explanted into agarose-coated culture wells. After three to five days, these microtumor fragments emerged as spheroids and could be maintained as organotypic spheroids for more than eight weeks. Measurements of the spheroids showed that they decreased during the initial two to three weeks and afterwards remained unaltered over a specific period of time. This growth pattern of the spheroids was consistent with the condition of tumors in vivo suggesting the linkage of cell proliferation and loss. Light microscopic and electron microscopic studies of the spheroids demonstrated that morphological structures were similar to those of the original tumor tissue in vivo and histopathologic characteristics of the original tumor were maintained over a long culture period. The spheroids contained connective tissues, blood vessels, macrophages, and neutrophils maintaining a three-dimensional architectural resemblance to the original tumors. Of three pairs of the surgical and spheroid specimen examined by the flow cytometry, one showed a change of ploidy pattern and two contained increased fractions of proliferating cells. It is concluded that this microtumor spheroid system can maintain the characteristics of the original tumors, and may serve as an alternative to the in vivo xenograft model for the research of brain tumor biology, invasion and immunology while providing a valuable technique for the evaluation of new therapies, such as biological response modifiers.