Measuring Global Cellular Matrix Metalloproteinase and Metabolic Activity in 3D Hydrogels

Abdulaziz S. Fakhouri; Jennifer L. Leight

doi:10.3791/59123

Method Article

Measuring Global Cellular Matrix Metalloproteinase and Metabolic Activity in 3D Hydrogels

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DOI:

10.3791/59123

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January 22nd, 2019

Abdulaziz S. Fakhouri¹^,²^,³ , Jennifer L. Leight¹^,²

¹Department of Biomedical Engineering, The Ohio State University, ²The Ohio State University Comprehensive Cancer Center, Arthur G. James Cancer Hospital and Richard J. Solove Research Institute, ³Biomedical Technology Department, King Saud University

Summary

Here, a protocol is presented for encapsulating and culturing cells in poly(ethylene glycol) (PEG) hydrogels functionalized with a fluorogenic matrix metalloproteinase (MMP)-degradable peptide. Cellular MMP and metabolic activity are measured directly from the hydrogel cultures using a standard microplate reader.

Abstract

Three-dimensional (3D) cell culture systems often more closely recapitulate in vivo cellular responses and functions than traditional two-dimensional (2D) culture systems. However, measurement of cell function in 3D culture is often more challenging. Many biological assays require retrieval of cellular material which can be difficult in 3D cultures. One way to address this challenge is to develop new materials that enable measurement of cell function within the material. Here, a method is presented for measurement of cellular matrix metalloproteinase (MMP) activity in 3D hydrogels in a 96-well format. In this system, a poly(ethylene glycol) (PEG) hydrogel is functionalized with a fluorogenic MMP cleavable sensor. Cellular MMP activity is proportional to fluorescence intensity and can be measured with a standard microplate reader. Miniaturization of this assay to a 96-well format reduced the time required for experimental set up by 50% and reagent usage by 80% per condition as compared to the previous 24-well version of the assay. This assay is also compatible with other measurements of cellular function. For example, a metabolic activity assay is demonstrated here, which can be conducted simultaneously with MMP activity measurements within the same hydrogel. The assay is demonstrated with human melanoma cells encapsulated across a range of cell seeding densities to determine the appropriate encapsulation density for the working range of the assay. After 24 h of cell encapsulation, MMP and metabolic activity readouts were proportional to cell seeding density. While the assay is demonstrated here with one fluorogenic degradable substrate, the assay and methodology could be adapted for a wide variety of hydrogel systems and other fluorescent sensors. Such an assay provides a practical, efficient and easily accessible 3D culturing platform for a wide variety of applications.

Introduction

Three-dimensional (3D) culture systems often more closely recapitulate in vivo cellular responses than traditional two-dimensional (2D) culture systems, see several excellent publications¹^,²^,³. However, utilizing 3D culture systems to measure cell function has been challenging due to the difficulty of cellular retrieval and further sample processing. This difficulty limits the measurement of many cellular functions in 3D culture systems. To overcome this difficulty, new techniques are needed that enable easy measurement of cell function within 3D environments. One wa....