Human disc cells in monolayer vs 3D culture: cell shape, division and matrix formation

Abstract

Background: The relationship between cell shape, proliferation, and extracellular matrix (ECM) production, important aspects of cell behavior, is examined in a little-studied cell type, the human annulus cell from the intervertebral disc, during monolayer vs three-dimensional (3D) culture.

Results: Three experimental studies showed that cells respond specifically to culture microenvironments by changes in cell shape, mitosis and ECM production: 1) Cell passages showed extensive immunohistochemical evidence of Type I and II collagens only in 3D culture. Chondroitin sulfate and keratan sulfate were abundant in both monolayer and 3D cultures. 2) Cells showed significantly greater proliferation in monolayer in the presence of platelet-derived growth factor compared to cells in 3D. 3) Cells on Matrigel(tm)-coated monolayer substrates became rounded and formed nodular colonies, a finding absent during monolayer growth.

Conclusions: The cell's in vivo interactions with the ECM can regulate shape, gene expression and other cell functions. The shape of the annulus cell changes markedly during life: the young, healthy disc contains spindle shaped cells and abundant collagen. With aging and degeneration, many cells assume a strikingly different appearance, become rounded and are surrounded by unusual accumulations of ECM products. In vitro manipulation of disc cells provides an experimental window for testing how disc cells from given individuals respond when they are grown in environments which direct cells to have either spindle- or rounded-shapes. In vitro assessment of the response of such cells to platelet-derived growth factor and to Matrigel(tm) showed a continued influence of cell shape even in the presence of a growth factor stimulus. These findings contribute new information to the important issue of the influence of cell shape on cell behavior.

Figure 1

Photomicrographs of ECM immunolocalizations of cells grown in the 3D microenvironment: Figures 1A and B are serial sections through a multi-celled colony evaluated for localization of chondroitin sulfate (Fig. 1A), keratan sulfate (Fig 1B). Figures 1C and D are serial sections through a multi-celled colony evaluated for localization of Type I collagen (Fig. 1C), and Type II collagen (Fig. 1D). Note positive black localization product for these ECM components. Figure 1E is a photomicrograph from a different specimen which was used as the negative control with deletion of primary antibody for this series of localizations with hematoxylin staining. (All photomicrographs X 295). Cells were from the same subject, were first passage and were grown for 10 days.

Figure 2

Photomicrographs of ECM localizations performed on whole cells grown in monolayer culture: For these studies, cells from the same subject were plated at the same density in 6 wells of a multi-well chamber slide and cultured for 10 days. Immunolocalizations were performed for each ECM product using a separate well. Prominent deposits of chondroitin sulfate (Fig. 2A) and keratan sulfate (Fig. 2B) can be seen surrounding the cells. In contrast, localizations for Type I collagen (Fig. 2C) and Type II collagen (Fig. 2D) show no deposition of these collagens by monolayer cultured cells. Figure 2E was taken from a well used as the negative control with deletion of primary antibody for these localizations with hematoxylin staining. (All photomicrographs, X 295).

Figure 3

Photomicrograph of the rounded cell phenotype and mounded colonies formed by cells growing on Matrigel™-coated plastic. This is a markedly different cell phenotype than is seen with growth on uncoated plastic as shown in Figure 2 (X 295).