Measurement of Aggregate Cohesion by Tissue Surface Tensiometry

Summary

We describe a method of measuring binding energy, expressible as tissue surface tension, between cells within 3D tissue-like aggregates. Differences in tissue surface tension have been demonstrated to correlate with invasiveness of lung, muscle, and brain tumors, and are fundamental determinants of establishing spatial relationships between different cell types.

Abstract

Rigorous measurement of intercellular binding energy can only be made using methods grounded in thermodynamic principles in systems at equilibrium. We have developed tissue surface tensiometry (TST) specifically to measure the surface free energy of interaction between cells. The biophysical concepts underlying TST have been previously described in detail^1,2. The method is based on the observation that mutually cohesive cells, if maintained in shaking culture, will spontaneously assemble into clusters. Over time, these clusters will round up to form spheres. This rounding-up behavior mimics the behavior characteristic of liquid systems. Intercellular binding energy is measured by compressing spherical aggregates between parallel plates in a custom-designed tissue surface tensiometer. The same mathematical equation used to measure the surface tension of a liquid droplet is used to measure surface tension of 3D tissue-like spherical aggregates. The cellular equivalent of liquid surface tension is intercellular binding energy, or more generally, tissue cohesivity. Previous studies from our laboratory have shown that tissue surface tension (1) predicts how two groups of embryonic cells will interact with one another^1-5, (2) can strongly influence the ability of tissues to interact with biomaterials⁶, (3) can be altered not only through direct manipulation of cadherin-based intercellular cohesion⁷, but also by manipulation of key ECM molecules such as FN^8-11 and 4) correlates with invasive potential of lung cancer¹², fibrosarcoma¹³, brain tumor¹⁴ and prostate tumor cell lines¹⁵. In this article we will describe the apparatus, detail the steps required to generate spheroids, to load the spheroids into the tensiometer chamber, to initiate aggregate compression, and to analyze and validate the tissue surface tension measurements generated.

Protocol

1. Aggregate preparation for measurement of tissue surface tension.

For adherent cells, spheroids can be formed by using either the hanging drop method or by generating a coherent sheet of cells that can then be cut into 1 mm fragments.

Aggregate formation by the hanging drop method:

1. Near-confluent adherent cell cultures should be grown to 90% confluence, whereupon monolayers should be rinsed twice with PBS. After draining well, add 2 mls (for 100 mm plates) of 0.05% trypsin-1 mM EDTA, and incubate at 37°C until cells detach. Stop trypsinization by adding 2 mls of complete medium and gently use a 5 ml pi....

Discussion

Measuring aggregate cohesion by TST is relatively straightforward. There are, however, key steps that must be mastered in order to generate useable TST data; 1) aggregates must be "healthy". This can be controlled by ensuring that aggregate formation begins with cells that are at optimal confluence prior to detachment. Aggregate size and time in culture must also be controlled to minimize the development of a necrotic core within the aggregate; 2) Another parameter that can influence TST measurements is the degree of adh.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
water bath/shaker	New Brunswick Scientific
10 ml round-bottom flasks	Bellco Glass