Unit 1 Topic 2 : cellular techniques

Cellular cultures

·       Most experiments require large populations of homogenous cells.

·       Consequently, there are many methods for maintaining cells in the lab.

·       Culture considerations :

-nutrients ( including gases): nutrient rich broth, (ex: female bovine system where cows get developing young, has a lot of amino acids, fats, sugars, etc.)

-diffusion of O2 to cells ; maintain certain level of density to have sufficient diffusion of gases in/out cell

-need to consider toxic compounds

-medium

- solid 2D medium, organized groups, steroids, etc.

-environment

-human cells typically require nutrient rich ( FBS) , solid media in a moist, warm environment

-temperature, moisture, submerged in fluid/ not submerged, how deep in media are cells kept?

primary cell cultures

·       cells prepared directly from tissues of an organism(already differentiated)

·       From tissue of organism

·       Has finite number of divisions

transformed cells

·     cells derived from a cancer or that have become oncogenic

-do not have finite number of divisions

stem cells

·       undifferentiated cells from growth tissue of an organism

·       Give rise to primary cells

·       Using stem cell instead of primary cell means have potential to propagate endlessly

embryonic stem cells

stem cells are derived from embryonic tissue

adult stem cells

-stem cells are derived from juvenile/adult tissue or tissues that support the fetus (umbilical cord, amniotic fluid, etc)

-reproduce to replenish blood, tissue, skin, nervous cells

-not pluripotent, but multipotent; could become multiple things

induced pluripotent stem cells (iPSC)

-stem cells (iPSC) are primary cells that have been induced to become undifferentiated

-through genetic manipulation take primary cells to undifferentiated cells

-downside : hard to get the cells to form what you want. Because they form anything, they do not settle into one thing

cell strains

·       derived from Primary cells

o   Differentiated

o   Limited number of divisions

o   Homogenous-because they are derived from one sample ; reduce noise in data, but limits how much you can from perspective of scientist. You can only say the culture works from the one cell

o   2D

cell lines

·      derived from transformed cells

o   Unlimited number of divisions

o   May not perform like original tissue: is there something they are doing that is giving false positive/negative?

o   Variable

o   2D

spheroids

·       3D organization of cell population

-usually 1-2 types of cell

-homogenous; not a lot variation

organoids

·       3D organization of multiple cell populations

·       Have some functionality of organ but doesn’t show what’s going on in other organs of the body

·       Not fully functional

·       Need to start with stem cells that produce 3D structures

·       More complexity of sample, realism, cost

Purification: Flow cytometry

·       Flow Cytometry can separate different cell types

 

o   Fluorescent antibodies are added to a cell mixture

o   As fluorescent-labeled cells are read, they are given a charge

o   Sorter separates cells by charge

o   Uses antibodies with fluorescent tags. Antibodies specific to protein on cell ; surface antigen

o   Purifies mixture to have individual cell types

 

·       Organelles can be separated using centrifugation.

o   Chemically (with detergents) or mechanically (sonification) lyse cells

o   Centrifuge

o   Add a sucrose solution and centrifuge more

o   Optionally purify with magnetic particles

Steps:

1.     Open cell ( chemically or mechanically) so they spill content

2.     Set up centrifugation to separate cells into different layers. If you want pure cells, can add super solution/sucrose because it is viscous and allows for easy sliding

3.     Spin it to separate organelles better

observing gene expression : Fluorescent in situ Hybridization (FISH)

·       Fluorescent complementary DNA sequences

·       Useful for finding where a gene is on a chromosome

·       Used for RNA sequences ( less stable) so risk losing fluorescence

observing gene expression: DNA microarrays

·       Thousands of oligonucleotides (short, 20-50 bp segments of DNA) are attached to a glass plate.  When fluorescently labeled DNA is poured onto the plate, they bind to the oligonucleotides, causing a fluorescent glow in the spot.

 

·       Plate with oligonucleotides. On other end there is a sample and what you do is label control with one fluorescent tag and treatment with other fluorescent tag

 

·       Oligonucleotides fish for sequence

o   Cluster analysis is a statistical method that uses the data from many microarrays.

Observing gene expression: RNA seq

·       Sequencing technique that copies every transcript from a sample (transcriptome)

o   Requires massive computational analysis

o   Several other methods have been developed using next generation sequencing (NGS) related to RNA Seq

manipulating gene expression: pharmaceutical perturbations

·       using drugs and/or enzymes to stop a particular cellular process; may have off-target effects

o   Ex: Cytochalasin A inhibits actin structures from polymerizing, and Brefeldin A inhibits Golgi functions

o   Great if you already know what pharmaceuticals will work

manipulating gene expression : genetic perturbations

·       altering gene expression to stop a particular cellular process

o   RNA interference (RNAi) –  using small interfering (siRNA) to block transcripts from being translated; difficult to get into cell

o   Effectively silence mRNA transcript

o   Create siRNA for particular gene wanting to silence

o   Have to shock cell in order for it to take in exogenous nucleic acid

o   Once nucleic acid is inside cell, it will silence transcript

o   Specific; target exact sequence looking for

o   Short lived

o   Want to keep silencing=keep applying

manipulating gene expression: CRISPR Cas9

o   replace a DNA segment with a specific variant, off-target effects

o   Protein that has some sequence in it that its trying to find

o   Like scissors, cuts sequence out + gobbles it up

o   Insert corrected sequence and let ends of chromosome cut itself back together

o   Problem: genomes are huge; so when dealing with crispr, it could edit a sequence somewhere else in a genome

o   Run sequence for long period of tine

manipulating gene expression: DNAzymes

o   catalytic sequence of DNA that degrade RNA; difficult to get into cell

o   DNA with enzymatic functions

o   Extremely specific; program what sequence you want to bind. Cleavage site in middle where RNA gets cut

o   Advantage over RNAi due to more stability