Culture Systems

Question 1

Culture conditions which can be controlled/minimized as an embryologist?

Answer 1

temperature
pH
osmolarity
air quality

Question 2

How are culture conditions optimized in the lab?

Answer 2

through:
- incubators
- culture media/oil
- protocols
- lab equipment
- training/skill of embryologist

Question 3

Why do we need to understand in vivo development of an embryo as embryologists

Answer 3

to replicate the environment in vitro to minimise embryo stress

Question 4

Key factors of in vivo environment to remember

Answer 4

temp = 37
pH= vag (4.4), fallopian tube/uterus (7.2-7.9) as embryologists we use 7.4-2.9
oxygen conc. = tube (5%), uterus (2%)
metabolic profile= oviduct (high pyruvate - TCA cycle), uterus (high glucose - glycolysis)

Question 5

Since the 2000’s what is the gas and humidification of incubators?

Answer 5

MINC incubators
- premixed gas bottles (89% Nitrogen, 6% CO2, 5% O2)
- charcoal filters used as humidifiers
- no sensors needed for when door open and shuts

Question 6

Since 2000 it was known that culturing embryos in low O2 environments is better than high O2 levels and 5% CO2. However this method was continuously used and in some labs are still cultured this way. Why is a high O2 environment not a good for the embryo?

Answer 6

impacts developmental competence
- causes oxidative damage and impacts function of mitochondria

• causes embryo to use glycolysis pathway over TCA cycle in the early cleavage stages
• linked to aneuploidy and mosaicism (in mouse embryos)
• generates excess of ROS

Question 7

How do the MINC incubators and how the embryos are set up in culturing helping reduce the stress of an embryo during culturing?

Answer 7

gas bottle (89% Nitrogen, 6% CO2, 5% O2) runs through charcoal filters and humidifiers
- cleans impurities in gas
- keeps temp at 37, oxygen environment low

embryos sit in:
- plastic dish
— not embryo toxic
- culture medium
— not enough = embryo flattened
—- shape of drops affect embryo outcome
- drops of media are covered with oil
— helps gas and temp exchange
— creates a more stable environment

Question 8

The embryos will spend 99% of their time in an incubator however there is inevitable stress when taking them out of the incubator due to atmospheric conditions. What are these conditions and what role does the embryologist play when the embryos are outside the incubator?

Answer 8

atmospheric conditions
- temp = 22-24
- oxygen = 21%
- carbon dioxide = less than 1%
- ambient air can contain VOC’s

embryologist must manipulate atmospheric conditions to reduce stress and be considerate of how often the incubator door is opened as the culture system is disrupted with atmospheric conditions

Question 9

How do temperatures too cold cause stress on the embryo?

Answer 9

below 37 cause a decrease in embryo viability as they struggle to maintain homeostasis

in oocytes:
- microtubules disrupted at 32 and may not reform even when back at 37
- some enzymes do not work efficiently at low temperatures

Question 10

How do temperatures above 37 degrees cause embryo stress?

Answer 10

maturing oocyte extremely sensitive to high temperatures (possibly unable to transcribe protecting proteins but unsure as to why)

embryos are sensitive to high temps but have the ability to transcribe own heat shock proteins HSP70

Question 11

How can we monitor temperature in the lab?

Answer 11

data loggers
- monitor fluctuations in all equipment
- need detailed logging and independent checks
- critical to measure exactly what the embryo will experience (ensure measuring from correct location)

MINC incubators reach 37 degrees in under 20 minutes while CO2 incubators over 3 hours

Question 11

Culture media is designed to have osmolality in the range of:

Answer 11

260-296

• if it is higher = water moves out of cells = embryo shrinks
• if it is lower = water moves into cells = embryo bursts

Question 12

How do time lapse incubators impact osmolality?

Answer 12

in time lapse incubators:
- dishes are more shallow
- drop size can be 10 fold smaller
- smaller oil layer also
- not humidified due to cameras

no humidification = drier air = higher evap because the air has ability to hold excess vapor when humidified air cannot hold more vapor therefore less evap

if there is more evap = decreases drop size = increases osmolality = embryo shrinkage

Question 13

What is the relationship between evaporation and osmolality and how does this affect lab procedures?

Answer 13

evap increases osmolality in culture media
- micro drops are high susceptible during preparation
- oil slows the evap but does not stop it
- all equipment for embryo manipulation is heated and promotes evaporation over time
- micro drops are not made on a warming stage for this reason
- less time on bench is better to avoid evap

Question 13

Historically culture media was made in house by embryologists based on human fallopian tube fluid. What was this media made up of?

Answer 13

patient serum
salts
antibiotics
metabolites
protien
pH indicator

Question 14

Due to the commercialisation of ART how is media made?

Answer 14

• a lot more complex
• patient serum is replaced by human serum albumin as it had components which were embryo toxic

Question 15

Why is sodium bicarbonate (NaHCO3) needed in culture media?

Answer 15

needed as a salt but also acts as a natural buffer in culture media

Question 16

How does NaHCO3 act as a buffer and how do we utilise this when creating media?

Answer 16

CO2 interacts with NaHCO3 and forms carbonic acid

Therefore when making culture media it needs to start at a higher pH (7.8-8) because in the incubator the NaCHO3 in the media will interact with the CO2 in the gas creating an acid which will lower the pH

Question 17

What colour should phenol red be at 7.2-7.4 range?

Answer 17

peachy apricot colour

Question 18

Bicarbonate buffer is required for which culture media and what needs to happen before it can be used?

Answer 18

fert media, cleavage media and blast media

made at a higher pH then needs to be exposed for a minimum of 4 hours to CO2 to shift the pH equilibrium to 7.2-7.4 before putting embryos in

Question 19

gametes, cleavage embryos and blastocysts all have different metabolic requirements. How is this reflected in culture media?

Answer 19

fert media
- high in glucose (sperm need high glucose to create ATP to swim)

cleavage media
- low glucose but high pyruvate (use TCA cycle at this stage)

blastocyst media
- high glucose low pyruvate (glycolysis being used as there is an increase in energy requirements)

Question 20

Embryos in culture have a high CO2 environment (5-6%) and require buffering with bicarbonate but on the bench we have low CO2 environments. What can it be buffered with on the bench?

Answer 20

buffered with HEPES or MOPS (zwitterion buffers and are stable in air)
- media still has NaOH but at low levels which do not have buffering capacity only acts as a salt

Question 21

Why can’t NaHCO3 be used has a buffer outside the incubator?

Answer 21

when bicarb is exposed to low CO2 (ambient levels) the pH increases to high levels within minutes outside the incubator

Question 22

How are HEPES or MOPS buffers stable at atmospheric CO2 (low levels)?

Answer 22

manufactured at 7.2-7.4 pH

this media still contains small amounts of bicarb so if put in the incubator will drop pH to toxic levels

Question 23

What types of media in the lab would contain HEPES or MOP buffers?

Answer 23

handling media
follicle flush buffer
gamete buffer

Question 24

What is phenol red, what is its role and is it found in all media?

Answer 24

ph indicator which can be added to some media for a visual measure of pH however most companies will avoid adding it to media due to its possible oestrogenic effects

Question 25

in vivo bicarbonate is present in the blood and plays a key role in embryo development. Is this the same for HEPES or MOPS?

Answer 25

no they alone do not support embryo growth and therefore should not be used for long time periods

Question 26

Why is high CO2 thought to be beneficial for embryos?

Answer 26

potentially need the excess carbon for key developmental processes

Question 27

What is sequential media?

Answer 27

two key media to address the changing metabolism of the embryo
- cleavage and blastocyst media

Clinic who use this media do a change over on day 3 @morulla / blastocyst stage

Question 28

What is single step media?

Answer 28

one media for whole culture period
- needed for time lapse incubators

can be done with or without change over
- without has the advantage of uninterrupted culture
- with has the advantage of less toxic build up

Question 29

How has commercialisation impacted embryologists in relation to culture media?

Answer 29

less awareness of what its media

Question 30

too much stress will have what impact on embryo?

Answer 30

epigenetic effects
lower chances of healthy offspring