Cell Bio Exam 1 Study Guide: Cell Separation, Fractionation, Protein Enrichment & Cell Culture, Tissue Engineering

Cell separation

Separating out a cell of interest in a tissue.

FACS

Fluorescence Activated Cell Sorting, a sophisticated system that can separate cells based on the association of the cell with a specific fluoresceinated tag.

Velocity Sedimentation

A cell separation system that relies on differential speed of cells through a gravitational field.

Selective Surfaces

Panning using beads/nanoparticles with selective surfaces that can bind to receptors of target cells.

GUAVA

$100,000 machine that can count fluorescently labeled cells in multiwell plates but does not separate them like a FACS.

Veridex CellSearch

FDA approved for counting CTCs, uses antibodies coupled to iron nanoparticles collected using magnetism.

Cell Fractionation

Lyse the cells and collect the organelle of interest.

Sonification

A method that destroys all organelles.

Detergent

Dissolves cells and destroys all organelles.

Trituration

Mechanical shearing that breaks open the cell, preferred for preserving organelles.

Differential centrifugation

A method used in protein purification.

Rough microsomes

Small bodies that originate from rough endoplasmic reticulum (RER) containing ribosomes.

Smooth microsomes

Small bodies that originate from smooth endoplasmic reticulum (SER) and contain plasma membranes and other organelle membranes.

EGTA

A calcium chelator that binds to calcium and disables Ca+2 dependent cell adhesion molecules.

Protease

Enzymes like Trypsin, collagenase, and liberase that clear proteins in order for cells to dissociate.

Edmorton Procedure

A procedure involving transplanted pancreatic islets for diabetes treatment.

Fluorochromes

Labels used to tag dissociated cells for FACS.

Iron core beads

Beads with a ligand that can bind to target cells and can be harvested using a magnet.

Anti-epCAM

An antibody specific to CTCs that is tagged with iron 'ferrofluid' for magnetic collection.

Magnetic matrix

Used in Veridex to pull out CTCs for counting.

Cell dissociation

The process of turning tissue into single cells.

Cell granularity

A property that can be used along with cell size for separation in FACS.

Density Gradient Centrifugation

Particles reach a point of equal density with the medium → then stop moving

Liquid chromatography

Column, beads → preserves the native activity of the proteins

Ion exchange chromatography

Elute (remove) the proteins using a sodium chloride (NaCl) gradient of 0 M NaCl → 1 M NaCl

Gel Filtration

Sizing column that uses a gel bead to separate proteins based on size and has beads with holes

Affinity chromatography

Technique used to purify proteins, such as the insulin receptor, which only fits into the specific binding site

Protein Purification

Enrichment of proteins using techniques such as Ion exchange chromatography

Tissue Dissociation

Process of treating tissue to end up with single cells, getting rid of other cell types and keeping the desired cell

Edmonton Procedure

Transplanted pancreatic islets to address diabetes, allowing patients to be insulin injection free

Veridex Cell Search system

FDA approved system for counting circulating cancer cells (CTC) using antibodies coupled to iron nanoparticles collected using magnetism

Cell Fractionation Techniques

Methods such as Sonication, Detergents, and Trituration used to break/lyse cells and collect organelles of interest

Sonication

Technique that destroys all organelles and can work with something in the cytoplasm

Detergents

Substances that dissolve cells and can work with something in the cytoplasm, destroying all organelles

Microsome

Small bodies derived from fragmented endoplasmic reticulum.

Smooth Microsome

Resealed outer cell membrane, involved in lipid synthesis.

Density Gradient Microfugation

A method where particles reach a point of equal density with the medium and stop moving.

Gel Electrophoresis

A technique that separates proteins based on molecular weight.

Native Gel Electrophoresis

Separates proteins on a polyacrylamide gel based on size and charge.

SDS Gel Electrophoresis

A method where proteins are denatured before separation, providing better resolution.

Isoelectric Focusing

Purifies proteins based on their specific isoelectric point using a pH gradient.

2-D Gel Electrophoresis

Combines isoelectric focusing and SDS gel electrophoresis for the best resolution.

Western Blot

A technique that combines gel electrophoresis and blotting to identify and quantify proteins using monoclonal antibodies.

Photoreactive Amino Acids

A method to create covalent bonds with proteins using UV light.

SELDI-TOF/MALDI-TOF

Mass spectrometry techniques that identify proteins with a resolution of 1 amino acid.

Protein A Immunoprecipitation

A technique that isolates proteins from staph aureus by binding to the Fc region of antibodies.

Autoradiography

Uses radioisotopes to track processes like gel electrophoresis.

Densitometry

A method that scans the density of a band and converts it to a number.

β-mercaptoethanol

A reducing agent that hydrolyzes disulfide bonds in proteins.

Urea

A compound that destroys hydrogen bonds in proteins.

SDS

A detergent that coats proteins, giving them an equal negative charge for separation.

Cell culture

Growing cells in an artificial medium (ex: bacteria, plant cells, yeast, mammalian cells)

In vitro

Cell culture

In vivo

In the living organism

In situ

In place

Golgi Stain

Can stain individual cells

HayFlick limit

Cell strains had a finite number of divisions then they would die

Cell strain

Freshly isolated from tissue with a limited number of divisions 25-50x (Hayflick limit)

Cell line

Immortal - keep dividing

Telomerase

Repairs telomeres every time they divide (ex. cancer cells)

Serum supplemented medium

Ex. 10% fetal bovine serum; contains growth factors

Defined medium

All is known - no FBS; critical for stem cell therapy

Microcarrier beads

Beads that can bind cells for large batches 'batch cultures'

Tissue engineered construct

Biological scaffolds that can be biodegradable or non-biodegradable

ELADS

Extracorporeal liver assist devices that take liver cells (not adult) and use C3A cells-hepatoma cell line

Carticel

Cartilage - autograft

Cardiac patches

Can these patches be used as a cardiac bandaid? Yes, clinical trials in US

Engineered human skin construct

Take cell culture dish with microporous membrane and put media in; increase concentration of extracellular calcium in the media to start differentiating

Neurospheres

Used to analyze vasculature

Stem cell therapy

A type of cell therapy

Gene therapy

A type of cell therapy

Regenerative medicine

Includes cell therapy, gene therapy, and tissue engineering

MAbs

Monoclonal antibodies which are made from Chinese hamster ovary cells and HeLa cells

Function can be lost over time

A limitation of cell culture

Genetic Drift

First seen in HeLa cells; true of all cancer cells

MDCK

Kidney cells, around since 1950; don't have a kidney-like function