BIEN190 Basics (should work)

What is the difference between a vertebrate and invertebrate?

Vertebrates have spines and invertebrates don’t

What is a primary cell line?

A cell line that has been taken directly from a patient

Write the flux equation

J = DdC/dx

What are two major differences between DNA and RNA?

DNA has thymine and deoxyribose and RNA has uracile and ribose

Is glycolysis an aerobic or anerobic process?

anerobic

The phase of bacterial growth that occurs at an exponential rate is known as?

log phase

What is a regulation?

A legally binding rule issued by a governing authority (a law), that defines how biological products are researched, produced, tested, released and monitored.

What is the consequence of not following a regulation?

fines, recalls, production halts, warning letters, criminal liability

What do regulations do?

Define what must be achieved and set minimum requirements

Are regulations mandatory or optional?

Mandatory

Are regulations written in broad or specific language?

broad, outcome focused language

Who makes and enforces regulations?

the federal government

What do regulations define?

safety, purity, potency, control expectations, documentation requirements, data integrity, legal responsibility of manufacturers

What is a standard?

Consensus-based technical specification

What does a standard define?

accepted methods, controls, and performance criteria used to meet regulatory requirements

Are standards optional or mandatory?

optional

What are consequences of not following a standard?

It is not illegal but you have to defend your alternative scientifically and statistically

Who makes standards?

ISO, ASTM, IEC

Are standards written in broad or specific language?

Highly technical and procedural

How do standards and regulations work together?

Regulations dictate the legal requirements for producing safe and effective biologics and standard provide the accepted technical pathway to meet those requirementsW

What does cGMP stand for?

current good manufacturing practice

What is a cGMP?

A regulatory term that means a compliance standard must reflect the current state of science and technology and are continually updated based on information available

What is the importance of a cGMP?

It prevents claims of ‘this was acceptable 20 years ago’

What happened to Genzyme?

There was viral contamination with Vesivirus 2117 in their mammalian cells (Chinese hamster ovary cells). Vesivirus causes skin blisters, its highly infectious and can last for months. This shut down the facility for a month because they were not following cGMPs but an older GMP, has insufficient in vitro cell culture controls, viral barrier and contamination prevention, and weak facility segregation and risk management. This virus was present for over a year prior but was not virulent to humans so no one died.

What happend to Tegenero TGN1412 Trail (2006)?

They are designed to stimulate T-Cells and attack leukemia and rheumatoid arthritis and directly stimulate the immune system. They did a phase 1 clinical trial that was double-blind, placebo-controlled, and had full disclosure of drug info, scientific info, and proper documentation. Within 90 minutes 6 healthy volunteers experienced catastrophic cytokin storm and had organ failure and long term effects. One person died. Found that if TGN142 was allowed to dry the molecular structure changed and resulted in a more potent mitogenic response than the positive controls. They should have tested on human blood more and their dose determination was flawed. They also followed GMPs and not cGMPs. Company was closed down.

What is cell culture?

Laboratory methods that enable growth of eukaryotic or prokaryotic cells in (or close to) physiological conditions or the removal of cells from an animal for plant and their subsequent growth in favorable artificial environment

What are the characteristics of bacteia?

prokaryotes, no nucleus, no membrane bound organelles

Are gram positive or gram negative bacteria more susceptibel to antibiotics?

gram positive

Describe 4 ways to grow bacteria?

Inoculation from frozen stock →colony pick from plate →overnight culture in test tube →experiment in Erlenmeyer flask

Inoculation from frozen stock →colony pick from plate →experiment in Erlenmeyer flask

Inoculation from frozen stock →overnight culture in test tube →experiment in Erlenmeyer flask

Inoculation from frozen stock →experiment in Erlenmeyer flask

What are some characteristics of yeast?

Eukaryote, great at fermentation reactions, used in a lot of food and pharmaceutical industries

What are the 2 ways that yeast grows?

Budding - new buds form off old cell and grow into new cell

Fission - original cell splits in half to create 2 cells

What are some common types of mammalian cells?

Chinese Hamster Ovary (CHO), Huamn Embryonic Kidney (HEK 293), Human Fibrosarcoma (HT-1080), HeLa (cerviacal cancer)

What are codons?

the building blocks of amino acids

What happens if a species chooses a codon it does not prefer (negative sign - on the chart)?

It will still make the protein but it will just be slower but this can also cause misfolding

What are some common cell culture considerations?

Nutrients and waste removal, growth, environmental controls, contamination monitoring, purpose, spatial constraints, time, mechanical forces

What are some nutrient and waste removal common considerations for cell culture?

the type of cell culture media

What are some common growth considerations for cell culture?

doubling time and passaging

What are some environmental control common considerations for cell culture?

O2, CO2, temperature, and humidity

What are some common contamination monitoring considerations for cell culture?

yeast, bacteria, virus, and mycoplasm

What are some common purpose considerations for cell culture?

production cell lines, cellular engineering, iPSC generation

What are some common spatial contraint considerations for cell culture?

volume and amount needed for experiments

What are some common time considerations for cell culture?

batch vs. continuous

What are some common mechanical force considerations for cell culture?

under conditions of flow, elastic modulus of surfaces

What are some common components of cell culture media?

water, salts to maintain osmolarity and membrane potential, nutrients (carbs, amino acids, minerals, growth factors, hormones), pH indicator, vitamins, trace elements (zinc, copper), and serum

What is serum?

The fluid that remains after clotting factors are removed from blood plasma. It is a concentrated solution of growth factors.

What is a common pH indicator?

phenol red

What can change the pH?

CO2

What is the downside of using phenol red?

It is an estrogen mimic in a lot of cells so it can change the behavior of cells

What is the downside of using serum?

There can be batch to batch variation

What are some serum examples?

Fetal bovine serum (FBS), Newborn calf serum, Horse serum

What is passaging?

The generation of a cell line and is also known as expansion/expanding. It is when cells are split up into more petri dishes to grow more cells

What is the most common way to get contamination during cell culture?

People not following aseptic technique

How do you treat for bacteria contamination?

antibiotics

How does bacteria contamination present in cell cultures?

The media will get cloudy and the pH indicator shows it getting acidic

How does yeast contamination present in cell cultures?

It presents similar to bacteria - cloudy and pH indicator shows its getting acidic

How do you treat yeast contamination?

Antimicotics

What are the indicators of mold contamination?

fuzzy stuff

How do you treat mold contamination?

throw out everything in the incubator and sterilize it

What are the indicators of mycoplasma contamination?

The pH indicator might be off, but it is very hard to determine if they are there

How do you treat mycoplasma contamination?

There is no way to treat

What is the indicator of a virus contamination in cell cultures?

A lower cell yield than expected

What are the benefits of using a primary cell line?

They are closer to what you would see physiologically to the source they came from

What is the downside to using a primary cell line?

passage is limited

What are finite cells?

Cells that divide a limited number of times

What are some characteristics of finite cells?

They are also known as passage limited and almost all primary cell lines are finite

What is senescence?

The phase cells have hit when they can no longer divide

What are immortalized cells?

Cells that can divide indefinitely

What is the downside of immortalized cells?

After a log of replication they will not behave the way the original cells do

What is morphology?

What the cell looks like which includes the shape, size, color, and surface to volume ratio

What is physiology?

What the cell can do which includes energy production, metabolism, biosynthesis, transport, cell division, protein production, homeostasis

You are comparing two eukaryotic cell types grown under identical conditions

Cell A has higher surface-area-to-volume ratio, an extensive endoplasmic reticulum, and a dense mitochondrial network

Cell B is larger, more spherical, has fewer mictochondria per unit volume, and a simplified internal membrane structure

Which cell would you expect to be more metabolically active? Briefly justify your choice using specific structural features.

Cell A because it has a dense mitochondrial network and high surface-area-to-volume ratio

You are comparing two eukaryotic cell types grown under identical conditions

Cell A has higher surface-area-to-volume ratio, an extensive endoplasmic reticulum, and a dense mitochondrial network

Cell B is larger, more spherical, has fewer mictochondria per unit volume, and a simplified internal membrane structure

Which organelle(s) most strongly support your conclusion (which cell is more metabolically active), and why?

Mitochondria because they produce energy in the form of ATP

You are comparing two eukaryotic cell types grown under identical conditions

Cell A has higher surface-area-to-volume ratio, an extensive endoplasmic reticulum, and a dense mitochondrial network

Cell B is larger, more spherical, has fewer mictochondria per unit volume, and a simplified internal membrane structure

If both cells were suddenly placed under low-oxygen conditions, which cell would experience functional failure first - and what structural limitation explains this?

Cell A because it has more mitochondria and needs more oxygen (aerobic process) to function. As it takes in oxygen it makes ROS (reactive oxygen species) which likes to bind to things (mostly CO2) and it will bind to anything it can. If you reduce oxygen you make more ROSs and they will start ripping away carbon from other stuff (membranes and DNA)

What are some characteristics of prokaryotes?

<10 um in size, no nucleus or membrane bound organelles, no cytoskeleton, singular chromosome, does not do meiosis/mitosis, has 70S ribosomes,

What are some examples of prokaryotes?

Bacteria, archae, blue-green algae, mycoplasma (bacteria without cell walls)

What are some characteristics of eukaryotes?

Membrane bound nucleus and organelles, they can have specialized areas of function, has a cytoskeleton, 10-100 um in size, has multiple chromosomes, does mitosis/meiosis, flagella are complex, are unicellular or multicellular, have 80S ribosomes

What are some characteristics of plant cells?

Have cell walls and vacuoles and usually has chloroplasts

What is the cell wall of plants made of?

Cellulose

What is the cell wall of fungi made of?

Chitin

What does the nucleus do?

stores DNA and chromatin

What does the Rough ER do?

protein synthesis

What does the Smooth ER do?

processes newly synthesized proteins and is the site of phospholipid synthesis and steriod synthesis and makes new cell membrane.

true or False. The Smooth ER is continuous with the nuclear envelope

True

Places in the body that make a lot of hormones will have a lot of Smooth or Rough ER?

Smooth ER

What does the Golgi Apparatus do?

Chemically modifies newly made proteins, packages proteins into vesicles that carry them to different locations. (almost all proteins that will be secreted will go through the golgi for further modification)

What do transport vesicles do?

Move things within the cell and between organelles

What do secretory vesicles do?

Move things to specific desitinations

What do lysosomes do?

They are specialized vesicles that contain a bunch of different enzymes (proteases, hydrogenases) and also have a lot of degratory enzymes (things that will break down molecules)

What is endocytosis?

Processes that move material into the cell by packaging it into a vesicle. It is ATP dependent (requires a lot of ATP) and does bulk transport into the cell. It is often involves a vesicle fusing with a lysosome

What is phagocytosis?

Type of endocytosis that takes in a large molecule

What is pinocytosis?

A type of endocytosis that takes in water

What is receptor mediated cytosis?

A type of endocytosis the allows the binding of specific molecules that can then be engulfed

What is exocytosis?

Transport of large molecules or particles out of a cell and often used secretory vesicles

What are some characteristics of mitochondria?

The site of ATP synthesis, will shut down if there is not enough oxygen, has 2 membranes (inner and external), the inner membrane has lots of folds (cristae) to increase surface area, the only organelle that has their own DNA, can grow and divide all on their own, and only comes from the mother (egg)

What does a chloroplast do?

They allow the plant to do photosynthesis and does carbon fixation (shoves CO2 together to make carbohydrates)

What does the cytoskeleton do?

Helps the cell keep its shape, allows the cell to migrate, and helps transport things within the cell

What are some characteristics of actin?

They have assembly and disassembly, they fuse together at z caps, and they are made on the positive side and degraded on the negative side

What are some things that make up the cytoskeleton?

Actin, microtubules, and intermediate filaments

What are some characteristics of microtubules?

They only geneate on the positive end and are a little larger than actin

What are some characteristics of dynein?

They load enzymes and proteins on microtubules, there is one that moves toward the exit and one that moves toward the center, and along with the movement of kinesin takes ATP