Preservation of glial cytoarchitecture from ex vivo human tumor and non-tumor cerebral cortical explants: A human model to study neurological diseases

Abstract

For the human brain, in vitro models that accurately represent what occurs in vivo are lacking. Organotypic models may be the closest parallel to human brain tissue outside of a live patient. However, this model has been limited primarily to rodent-derived tissue. We present an organotypic model to maintain intraoperatively collected human tumor and non-tumor explants ex vivo for a prolonged period of time ( approximately 11 days) without any significant changes to the tissue cytoarchitecture as evidenced through immunohistochemistry and electron microscopy analyses. The ability to establish and reliably predict the cytoarchitectural changes that occur with time in an organotypic model of tumor and non-tumor human brain tissue has several potential applications including the study of cell migration on actual tissue matrix, drug toxicity on neural tissue and pharmacological treatment for brain cancers, among others.

Figure 1

Evaluation of normal adult human cortex obtained from a 46 year old female using electron microscopy (EM) and immuohistochemistry (IHC). a,e,i, 0 days in culture. b,f,i, 3 days in culture. c,g,k, 7 days in culture. d,h,l, 11 days in culture. The explants retained their cytoarchitecture for at least 11 days while in culture as seen by both EM and IHC. With the use of IHC analysis, the tissue cytoarchitecture remained intact through 11 days of culturing. This was evident by the numerous astrocytes and intact astrocytic processes. (a-d; GFAP-green, DAPI-blue). With the use of EM analysis, glial bodies appeared to persist through 11 days of culturing, and large vacuoles were notably absent, signifying surviving cells (e-h). At higher magnification, the cell membranes are maintained without distortion and the synaptic contacts are highly preserved (i-l). Moreover, both the presynaptic and postsynaptic vesicles can be clearly seen, as well as mitochondria (l: arrows) without altered morphology (e-h).

Figure 2

Non-tumor and Tumor Organotypic Explants at 14 days. A, Non-tumor cortical explant at 14 days displaying very little DAPI (nuclear marker) staining and sparse astrocytic processes. B, Glioblastoma tumor explant at 14 days displaying very few DAPI staining and infrequent astrocytic processes.

Figure 3

Evaluation of human glioblastoma multiforme obtained from a 55 year old male using electron microscopy (EM) and immuohistochemistry (IHC). a,e,i, 0 days in culture. b,f,j, 4 days in culture. c,g,k, 6 days in culture. d,h,l, 10 days in culture. Unlike the normal cortex, the tumor cytoarchitecture appeared less organized and thus more characteristic of tumors (a-d; GFAP-green, DAPI-blue). The tumor explants retained their cytoarchitecture for at least 10 days while in culture as seen by both EM and IHC. With the use of EM analysis, the organization of the tumor cells in culture did not show any significant differences compared to tumor cells observed in vivo (e-h). At higher magnification, the characteristic organelles within the tumor astrocytes were preserved. This included intermediate filaments (i, l: arrows) and rough endoplasmic reticulum (j: arrows). Mitotic bodies were also seen in the explants, supporting the notion that the tumor explants were still proliferating and were remarkably preserved (k) (e-h). With the use of IHC analysis, the tissue cytoarchitecture from the tumor explants remained intact through 10 days of culturing.

Figure 4

EM analysis of neurons at 3 days. In contrast with glial cells that were well conserved with prolonged culturing, the neurons degenerate rapidly (A). Intact neurons were infrequently observed (B).

Figure 5

EM analysis of normal human cortex under the standard protocol used for mice (Stoppini et al. 1991; Tanaka et al. 1994). A, 7 days in culture. B, 14 days in culture. Under these conditions, the cytoarchitecture of the explants were not preserved even at 7 days. With continued culturing, less cell bodies were observed, membranes appeared more diffuse and irregular, and synaptic contacts and vesicles were less infrequently observed as compared with the revised protocol.