Biological Sciences 121 Outline 3
Ewing
Studying Cells In Vitro
I. Cell physiologists and cell biologists use cultured cells extensively to study cellular processes. These cultures are called in vitro (in glass) cultures as compared to cells in vivo: cells in place in the living organism. The terms in vitro and in vivo have different meanings to biochemists where in vivo refers to the study of a process in a living cell or organism regardless of how the cell is grown. For example, we could study the activity of an enzyme in a cultured cell—this would be referred to as an in vivo analysis. If the cell were now disrupted and the enzyme was studied in a test tube—this would be an in vitro analysis.
A. Many classes of cells can be cultured. Those that can be cultured most easily have been studied most extensively. These provide excellent systems for studying processes that would be difficult to approach in a living organism.
1. many types of animal cells will form a monolayer when cultured in a dish with the appropriate surface with the proper signal molecules
a. studying cells in culture has allowed many of these key signals to be discovered
b. early work showed that serum (clotted blood from which red blood cells have been removed) could provide necessary signals.
c. it then became a hunt to discover what those signals were. These signals were discovered and are referrred to as growth factors.
2. Cells can be isolated form specific tissues to generate homogeneous cultures. These usually die after a limited number of divisions. Occasionally cells undergo a change that allows them to continue to grow. These are referred to as cell lines. Specific cell lines have been maintained and passed from lab to lab.
3. Examples of cell lines we will see often throughout this course include:
a. epithelial cells: form a monolayer. Intestinal epithelial cells will grow with microvilli upward and tight junctions intact. Appear to closely resemble epithelial cells in vivo.
b. growing neurons: allow signals that guide growth to be tested (in lab we’ll use pollen tubes to mimic this even more simply)
c. fibroblasts: many uses, including studies that examine cellular locomotion and cell adhesion
II. Subcellular Fractionation
A. To simplify the study of cellular processes cell physiologists and cell biologists have also made extensive use of subcellular fractionation.
1. This is the disruption of cells followed by the separation of cellular components. Processes may be studied in crude preparations or in increasing pure preparations resulting from sequential purification steps.
2. differential centrifugation is a rapid and effective technique for separating cellular components. We will use this in the lab to isolate mitochondria. We will then study cellular respiration in these isolated mitochondria exactly as was done to unravel the mechanism of respiration.
3. many additional techniques can be applied to isolate, purify and analyze cellular components. SDS-PAGE (polyacrylamide gel electrophoresis) was a huge breakthrough for studying proteins, especially membrane proteins. Allows proteins to be separated by size and visualized. (This is covered fairly extensively in the reading—just try to get a feel for potential uses here.)
a. we will often refer to the sizes of individual proteins. In many cases these are first determined by purifying the protein and subjecting it to SDS-PAGE.
b. to define the amino acid sequence of the protein it is easiest to clone the gene that encodes the protein, determine its DNA sequence and predict the sequence of the protein it encodes
c. from the amino acid sequence of the protein we can often predict more about its structure and function.
d. purified proteins can be crystallized and their exact structure determined using X-ray crystallography
III. Cell-Free Systems
A. To define the key elements in a process cell physiologists and cell biologists make use of cell-free systems. Here, purified components are combined in attempts to reconstitute cellular processes. Depending upon the purity of the components added, these studies can allow the minimum components necessary for a process to be defined. This is a key step in beginning to fully understand a process.