Cell Biology: Centrifugation and Cell-Free Systems

Cell Biology

• Cells are the basic morphological and functional units of the body, and are too small to be seen with the naked eye.
• Development of cell biology had to await the development of a magnifying device, such as the light microscope.
• The term "Cellula" was first used by Robert Hook in 1665 to describe small chamber-like structures observed in cork under a microscope.

Cell Components

• Protoplasm is the living substance of plants and animals, comprising the nuclear region and cytoplasm.
• The smallest unit of protoplasm capable of independent existence is the cell.

Cell Theory

• Cells are the fundamental units of both structure and function in all living organisms.
• All living organisms are composed of cells and their products.
• Cells arise only from preexisting cells.

Cell Culture and Applications

• Microdissection techniques allow selected cells to be isolated from tissue slices.
• Applications of cell culture include:
  • Embryonic organs can be cultured.
  • Study of cancer cells.
  • Cell to virus relations.
  • Cytogenetic research.
  • Cell-cell interactions.
  • Cell nutrition.

Stem Cells

• Embryonic stem cells are the most promising cell lines from a medical point of view.
• These cells can proliferate indefinitely and give rise to all cell types in the body.
• ES cells might be used to replace and repair damaged mature human tissues.

Model Organisms

• Escherichia coli is the best understood cell in biology.
• Saccharomyces cerevisiae (yeast) is the most frequently used single cell eukaryote.
• Studies with bacteria and yeast established the basic principles of molecular biology.

Cell Fusion

• Cell fusion is the process of membrane merging and cytoplasmic mixing of two cell types.
• Hybrid cells are produced by fusion of animal cells.
• Examples include human cell and mouse cell fusion.

Microdissection Techniques

• Allow selected cells to be isolated from tissue slices
• Applications include:
  • Embryonic organ culture
  • Study of cancer cells
  • Cell-virus relations
  • Cytogenetic research
  • Cell-cell interactions
  • Cell nutrition

Embryonic Stem Cells

• Most promising cell lines for medical applications
• Derived from inner cell mass of early mouse embryos
• Can proliferate indefinitely
• Can give rise to all cell types in the body
• Can be used to replace and repair damaged mature human tissues
• Potential therapeutic applications:
  • Muscular dystrophy
  • Parkinson's disease
  • Type I diabetes

Model Organisms

• Escherichia coli (E. coli): best understood cell in biology
• Saccharomyces cerevisiae (yeast): most frequently used single-cell eukaryote
• Studies with bacteria and yeast established basic principles of molecular biology

Cell-Cell Fusion

• Hybrid cells produced by fusion of animal cells
• Cell fusion: membrane merging and cytoplasmic mixing of two cell types
• Examples: human cell and mouse cell fusion

Cell Size and Number

• Human body composed of trillions of cells
• Size of a typical animal cell: 20-30 µm
• Largest cell: human egg (150-200 µm)
• Smallest cell: granular cells of cerebellum (4-5 µm)

Methods for Examining Cells

• Examination of the cell as a whole (without disruption)
• Examination of living cells (fresh tissues)
• Examination of killed and preserved tissues and cells
• Fractionation of cells and analyzing their molecules

Cell Culture

• Isolating cells and growing them in culture
• Living cells can be suspended in an appropriate liquid (e.g. saline solution)
• Prolonged study of living cells can be made by culturing them in solutions containing necessary nutrients
• First tissue culture (1907): Ross Harrison, isolated fragments of spinal cord of a frog

Culture Conditions

• Medium supplying essential nutrients (amino acids, carbohydrates, vitamins, minerals)
• Growth factors
• Hormones
• Gases (O2, CO2)
• Regulated physico-chemical environment (pH, osmotic pressure, temperature)
• Most cells are anchorage-dependent and require a solid or semi-solid substrate

Primary Cell Culture and Cell Lines

• Primary cell culture: derived from normal animal cells, limited life span, divide 25-50 times, and then stop dividing (cell senescence)
• Cell lines: eukaryotic cell lines, widely used for experiments, transformed cells, grow indefinitely in culture (immortal cells)

Fractionation of Cells and Analyzing their Molecules

• Cell fractionation is a method to study certain organelles from a cell, involving the disruption of cells and isolation of organelles and macromolecules in pure form.
• Cells can be disrupted in various ways, such as osmotic shock, ultrasonic vibration, or grinding up.

Cell Disruption Methods

• Chemical: alkali, organic solvents, detergents
• Enzymatic: lysozyme, chitinase
• Physical: osmotic shock, freeze/thaw
• Mechanical: sonication, homogenization, French press

Steps of Subcellular Fractionation

• Homogenization
• Differential centrifugation
• Further separation and purification by density gradient centrifugation
• Collection of fractions
• Analysis of fractions

Centrifugation

• Centrifugation is the first step in most fractionations, separating components that differ greatly in size.
• Low speed centrifugation is used to separate intact cells from medium.
• High speed centrifugation can be used to separate subcellular components.
• When a centrifugal force is applied to an aqueous mixture, components of larger size and density will sediment faster.

Density Gradient Centrifugation

• A finer degree of separation can be achieved by layering the homogenate as an arrow band on top of a dilute salt solution that fills a centrifuge tube.
• The various components in the mixture move as a series of distinct bands through the salt solution, each at a different rate, in a process called velocity sedimentation.

History of Mitochondria Isolation

• 1934: First isolation of mitochondria from liver by Bensley
• 1937: First chemical analysis of mitochondria

Isolation of Components of Living Cells by Differential Centrifugation

• Organelles and macromolecules can be separated by ultracentrifugation.
• Pure fractions of nucleus, nucleolus, mitochondria, lysosomes, microsomes, and ribosomes can be isolated.
• This method has made it possible to obtain knowledge on the molecular composition of cell components.

Method of Differential Centrifugation

• Cut tissue in an ice-cold isotonic buffer to stop enzyme reactions, osmosis, and pH changes.
• Grind tissue in a blender to break open cells.
• Filter to remove insoluble tissue.
• Centrifuge filtrate at low speeds (1000 X g for 10mins) to pellet nuclei.
• Centrifuge at medium speeds (10 000 x g for 30 mins) to pellet mitochondria.