With the advent of highly sensitive molecular analysis technologies, it is possible to study increasingly complex cellular processes in diseases like cancer. However, the tissue sample consists of different cell types, in different states. Therefore, the ability to analyze and separate a heterogeneous cell population based on cellular characteristics is important for molecular analysis techniques. The cellular components of tissue are bonded tightly together by extracellular material (ECM). The ECM contains proteins, which are broken down by enzymes like metalloproteinases. Though the enzyme dissociation process is an integral part of life sciences laboratory, it is mainly designed empirically. It can be predicted and improved by applying fundamentals of enzyme kinetics of ECM degradation and diffusion of enzyme molecule through ECM. This research utilized breast tissue (normal and malignant) as a model, but the principles are universal.
This study focused on understanding each ECM component and concluded that collagen concentration and distribution are the rate controlling component in breast tissue digestion. It was hypothesized that actual time of tissue digestion can be modeled based on digestion time of collagen. Primary variables like collagen concentration, kinetics of collagen degradation and diffusion of collagenase in the breast tissue were studied. It was quantified that average collagen concentration in breast tissue is 53 ± 37 µg of collagen per mg of wet breast tissue. The kinetic parameters of collagenase was quantified based on fluorescence measurement as 5.9 ± 3.1 µg of collagen/U.min (kcat) and 7.2 ± 1.7 µg of collagen/ml (KM). These kinetic parameters along with the collagen concentration were utilized to calculate the time of digestion based on Michaelis – Menten kinetics. This time was an order of magnitude less than observed by researchers, which ranged from 1 to 16 hours for breast tissue. This discrepancy in the time of digestion was attributed to mass transfer of enzyme in the tissue matrix. The diffusion of enzyme was modeled based simple reaction diffusion model and the predictions were confirmed with empirical studies. Based on these results, it was confirmed that time of breast tissue digestion can be predicted based on time of collagen digestion.