Cell culture and harvest

Question 1

Basic media

Answer 1

Media that contains only the essential support elements for cell culture. These include amino acids, vitamins, minerals and glucose.

Question 2

Modified media

Answer 2

Media that has been modified to contain a support element that enhances the growth of a particular cell type. For example selenium can be added to media to enhance the growth of breast cancer cells

Question 3

Enriched media

Answer 3

Media that contains extra ingredients that will encourage cell growth. For example giant cell tumor conditioning media can be added to basic media to enhance the growth of leukemic cells

Question 4

Complete media

Answer 4

Media that contains everything that cells need to grow in culture. This includes L-glutamine and fetal calf serum

Question 5

Maintenance media

Answer 5

Media in which the amount of fetal calf serum has been reduced to the point that cells remain viable, but their growth rate is slowed

Question 6

Ham’s F10 media

Answer 6

1% L-Glutamine
1%Vitamins
0.5% Pen/Strep
10% GCT(Giant cell tumor conditioning media) IGen
10% Fetal calf serum

Question 7

Buffers

Answer 7

A substance capable in solution of neutralizing both acids and bases and thereby maintaining the original acidity or basicity of the solution

Question 8

Commonly used buffers

Answer 8

1. Sodium bicarbonate reacts with hydrogen ions to form water, carbon dioxide and sodium carbonate and thereby can act to buffer metabolic acidosis. Sodium carbonate has a higher pH than sodium bicarbonate and it is this byproduct that buffers the media.
2. HEPES - organic buffer

Buffers are used in cell culture media to maintain a constant pH. Physiological range for pH is 6.6 - 7.8; Optimal pH range for cell culture is 7.2 - 7.4.

Question 9

ph indicator

Answer 9

phenol red
pH 8.0 magenta color
pH 7.2 - 7.4 red-orange color
pH 6.8 or less yellow color

Question 10

PHA

Answer 10

T cells

Question 11

LPS

Answer 11

B cells

Question 12

PWM - Pokeweed

Answer 12

T & B cells

Question 13

CLL and B-cell lymphoma

Answer 13

24hrs & 72 hrs LPS

Question 14

T-cell lymphoma

Answer 14

24 hr & 72 hr PHA

Question 15

All others

Answer 15

24&48 hrs

Question 16

PHA

Answer 16

at 24 hrs: T lymphocytes will show an increase in RNA synthesis signaling a transformation of lymphocytes into lymphoblasts.
at 48 hrs: Most of the mitoses observed are the result of one cell division.
at 72 hrs: Most cells have undergone two cell divisions.

Question 17

Chromosome breakage syndromes

Answer 17

Fanconi’s anemia
Bloom’s syndrome
Ataxia Talangiectasia
Xeroderma Pigmentosum

Question 18

Fanconi’s anemia

Answer 18

sensitive to : DNA crosslinking (alkalyating) agents, UV light and ionizing radiation
DNA repair deficiency: Abnormal activity of topoisomerase and DNA ligase l activity decreased
clastogen used in culture: Mitomycin C

Question 19

Bloom’s syndrome

Answer 19

sensitive to:UV light and ionizing radiation
DNA repair deficiency: DNA ligase I
clastogen used in culture: none; spontaneous

Question 20

Ataxia Talangiectasia

Answer 20

sensitive to:ionizing radiation: X-rays and gamma radiation
DNA repair deficiency:Polymerase
clastogen used in culture:Bleomycin or other radiomimetic drugs

Question 21

Xeroderma Pigmentosum

Answer 21

sensitive to:UV light
DNA repair deficiency:Excision repair
clastogen used in culture:UV mimetic drugs such as 4NQO

Question 22

Prophase cultures

Answer 22

Ethidium bromide, Actinomycin D, BrdU reduce the contraction of chromosomes during these stages of the cell cycle: G2, M
MTX(amethopterin), FudR, excess Thymidine react with cells at these stages of the cycle: S

Question 23

Fragile X syndrome

Answer 23

This genetic disorder is characterized by an expansion of triple repeats of CGG nucleotides near the 5’ end of the FMR1 gene located at band Xq27.3.
6 – 60 repeats – normal phenotype
60-200 repeats – premutation carrier
>200 repeats – Fragile X phenotype
Folate deficient/thymidine deficient media, e.g. TC 199 or MEM-FA.

Question 24

BM culture minimum cell requirement

Answer 24

minimum 1 x 10^6 cells/ml in each culture

Question 25

Incubation period

Answer 25

1. Direct
2. Short term
3. Long term

Question 26

direct preparations

Answer 26

+: Quick analysis

-: poor yield, inferior quality of metaphases

Question 27

short term

Answer 27

+: Most successful technique

-: Culture dependent, clonal selection

Question 28

long term

Answer 28

+: Established cell lines, good quality
- : Stromal overgrowth,
in vitro artifacts

Question 29

Aseptic culture techniques

Answer 29

1. Prevent microbial contamination

2. Prevent cross-contamination between cultures

Question 30

Aseptic technique

Answer 30

Wipe down the surface of the hood with 70% ETOH prior to use
Do not block the front vent by placing objects over the grille.
Do not pace your hand over any opened bottle or culture flask.
Wipe down the surface of the hood with 70% ETOH after each use.

Question 31

Horizontal laminar flow hood

Answer 31

Protects only the specimen from contamination

Question 32

Class I Biological Safety Cabinet

Answer 32

Protects only the worker from contamination

Question 33

Class IIA &B Biological Safety Cabinet

Answer 33

Protects both the specimen and worker from contamination

Question 34

Class III Biological Safety Cabinet

Answer 34

Protects both the specimen and worker from contamination

Question 35

Mycoplasma Contamination

Answer 35

Mycoplasma or PPLO (pleuropneumonia-like organism) are the smallest free-living organisms which have been identified and have been found to retard growth and induce karyotypic changes in long term cultures. Mycoplasma are so small that they can’t be observed under the inverted scope. Cultures suspected to be contaminated must be stained with DAPI or Hoechst 33258 in order to confirm mycoplasma contamination. BM cycline is the most effective antibiotic for controlling mycoplasma (final concentration 5ug/ml)

Question 36

Antibiotics

Answer 36

Pen/Strep : bacteria only
Gentamycin: yes for all(bact; yeast/fungi; mycoplasma)
Fungizone (Amphotericin B): fungi/yeast & mycoplasma
Mycostatin: only mycoplasma

Question 37

If contamination is detected

Answer 37

Incubate media and check for a visible change in color or turbidity or the presence of microbes when viewed under the microscope.
Check any other solution that is added to the media or culture such as: trypsin, or serum.
To prevent spreading contamination to other cell cultures, work with only one sample at a time in the hood and maintain separate media bottles.

Question 38

Prevent Cross-Contamination

Answer 38

Maintain separate media, trypsin, and Hank’s for each culture
Use a new pipette for each different cell culture/specimen.
Work with one culture in the hood at a time
Long term cell lines can easily outgrow and take over primary cultures.
Also fibroblasts cells can overtake slower growing epithelial cells.

Question 39

Cultural Maintenance

Answer 39

Maintain proper temperature

Incubator temperature should be set and maintained within a very limited range (36-38oC.) Lower and higher temperatures can adversely affect cell growth. This is within the temperature range of most warm-blooded animals. When the temperature rises over 40oC, cells will soon die.

Maintain proper CO2 level

In open systems 5% CO2 is required and the chamber is often humidified to prevent the evaporation of the media. Some media require higher CO2 levels to maintain proper pH. For example, DMEM requires a CO2 level of 10%.
Feed cultures at regular intervals
Subculturing

Subculturing is the process of removing cells from one culture vessel and transferring only a proportion of the original cells into new culture vessels. This process is also commonly called “splitting” the cultures. When cell cultures are composed of actively dividing cells they can be subcultured relatively thin (1:3 or 1:4.) Slow-growing cultures should be subcultured relatively thick to encourage cell growth.

• suspension cultures - To subculture these cells, one has only to centrifuge the culture contents, discard the used media, then resuspend the cell pellet in fresh media and distribute the cells into new culture vessels.
• monolayer cultures - To subculture these cells, one has to remove the cells from the bottom of the culture vessel. Trypsin is commonly used (concentration ranges from 0.01% to 1.25% have been reported.) EDTA is added to trypsin when difficulty in detaching the cells from the flask is encountered. EDTA is a chelating agent. Chelating agents bind loose calcium ions which can bind to the active site of trypsin. Removing these ions can dramatically improve the effectiveness of the tissue culture trypsin.

Question 40

Cryopreservation

Answer 40

Glycerol - 5% (30 minute treatment)
DMSO - 10% (10 minute treatment)
Dehydration of cell prevents formation of ice crystals
Cell density important factor
-90oC for one year or -195oC for extended storage

Question 41

Cryopreservation procedure

Answer 41

1. Select for freezing - Cell density should not be low or too high to ensure successful thawing in the future. These chemicals dehydrate the cell preventing the formation of ice crystals, which can rupture the cell membrane upon thawing. If cells are too many, cells, which settle out, will not get enough exposure. If cells are too few, there will not be enough viable cells after thawing to initiate a culture.
2. Freeze cells

Single cells should be resuspended in one of the following solutions:

Glycerol - 5% concentration in complete media
DMSO (Dimethyl sulfoxide) - 10% concentration in complete media
After approximately 30 minutes of treatment:

Cells can be placed into a freezer, which manually lowers the temperature 1 - 3oC each minute before placing in a nitrogen freezer.
Cells can be stored in -90oC freezer for up to one year.
Cells may be immediately stored in a nitrogen freezer at -195oC
3. Record data in log - All frozen specimens should be logged into a logbook and assigned an accession number. Information such as patient name, passage number, freezing method, freezer location (box, column and row information) and cell density should be recorded for future reference. All freezer vials should be clearly labeled with patient reference number or name and log accession number.

Question 42

Maintain Cell Pellet for Long Term Use

Answer 42

Cells fixed in 3:1 Carnoy’s fixative can be stored at –20°C for several years. Cells should be stored in a polypropylene container that is closed tightly to prevent evaporation. When saved suspension over one week old is used to drop new slides, the cell suspension should be centrifuged and the old fixative replaced with fresh fixative.

Question 43

No growth troubleshooting

Answer 43

Change the media
Try a new bottle or lot of media.
Freshen up old media by adding a little additional L-Glutamine or Fetal Calf Serum
Try a different brand of media.
Plasticware may be toxic or contain residues slowing cell growth. Try a new lot number. If cells are not attaching to the bottom of flask in monolayer cultures, centrifuge and reduce the amount of media

Question 44

Single cell suspensions

Answer 44

These cultures are the easiest to harvest, for they can easily be transferred to a tube where the harvest solutions can readily be added and removed.

Question 45

Monolayer flask cultures

Answer 45

When working with these types of cultures it is necessary to first remove the cells from the flask by using either a trypsin/EDTA solution or a cell scraper. This will dissociate the monolayer of cells into a single cell suspension. This suspension should then be transferred to a tube and processed the same as for single cell suspensions.

Question 46

In situ culture

Answer 46

Cells grown on coverslips or in slide chambers are harvested within the culture vessel itself. Throughout this harvesting procedure, cells remain attached to the surface of the coverslip or slide. This has advantages in those cases where the number of cells available for culture is low and in those cases where the clonality of cytogenetically abnormal cells provides important clinical information

Question 47

Elongation Techniques

Answer 47

Prior to the harvest procedure, various chemicals may be added to the cultures, which can either interfere directly with the contraction of metaphase chromosomes or indirectly allow collection of a large number of cells in prophase where the chromosomes are elongated.
Synchronization agents
DNA intercalators

Question 48

Synchronization agents

Answer 48

These chemicals block cell division at the G1/S border by blocking the formation of nucleotides needed for DNA synthesis.

Question 49

DNA intercalators

Answer 49

chemicals, which can enter the nucleus of a cell and can then insert themselves between the nucleotide bases. The insertion of these molecules between the nucleotide bases interferes with the process of chromosome contraction, which occurs during the metaphase period of the cell cycle. Because of this ability, they are in all cases genotoxic and must be handled with the use of gloves and without breathing the dust.

Question 50

Ethidium bromide

Answer 50

An intercalating agent that is thought to prolong the period between prophase and metaphase by binding certain proteins responsible for chromosome condensation during mitosis.

Question 51

Actinomycin D

Answer 51

Preferentially binds to guanine residues during the G2 period of the cell cycle and prevents the protein binding needed for chromosome condensation

Question 52

BrdU

Answer 52

a thymine analog interferes with the normal condensation of the DNA helix at the tertiary level.

Question 53

DAPI, Hoechst 33258 and Distamycin A

Answer 53

DNA binding agents which specifically cause the heterochromatic regions of metaphase chromosomes to remain undercondensed as compared to the euchromatic regions of metaphase chromosomes.

Question 54

Mitotic Inhibitors

Answer 54

Mitotic inhibitors trap cells in metaphase. Universally they depolymerize the microtubules that make up the spindle apparatus. With the spindle inactivated, the cell cannot undergo cytokinesis or cell division.
When cells leave G2 and enter prophase, there are proteins, which bind to the DNA and induce chromosome condensation. Maximum chromosome compaction enables the cells to divide and split with the minimum of errors such as anaphase bridges and lagging chromosomes. The chromosomes are maximally condensed by late metaphase.
Due to the two factors above, if a cell is arrested in metaphase, the number of metaphases you can trap and the contraction of the chromosomes is a function of time. If one were to assume that mitosis lasts 15 minutes, mitotic arrestants added for 1 hour to the culture prior to harvest would result in approximately 4 times the number of metaphases. One-fourth of these metaphases will be of the normal length, 1/4 will be twice as short as the normal metaphase length, 1/4 will be three times as short as the normal metaphase length, and 1/4 will be four times as short as the normal metaphase length. (Remember that normal metaphase length is a range from early prophase to late metaphase).
The Stages of Mitosis

Mitotic arrestants themselves also cause the rate of chromosome condensation to increase. Reducing the concentration to 1/4 of the usual dose will allow cells to be arrested in metaphase without becoming extremely short.
Colchicine
Colcemid - This analog of Colchicine is widely used. It is less toxic than Colchicine. In fact replacing the Colcemid containing media with media without Colcemid can reverse this mitotic block.
Vincristine (Velban)

Question 55

Hypotonic Solutions

Answer 55

Passive transport allows the crossing of H20 across the cellular membrane. This is an ATP independent process, and equilibrium is determined by the salt concentration within and outside of the cell. In the test tube it is possible to manipulate the salt concentration of the environment. Hypotonic solutions cause water to rush inside the cell at a greater rate than would normally occur. Hypertonic solutions have the opposite effect. Passive transport is different from active transport that is an ATP dependent process. When live cells are subject to hyper or hypotonic environments, active transport is what the living cell uses to counteract an abnormal osmotic pressure and maintain the normal cell water/salt equilibrium.
Prior to mitosis the nuclear membrane dissolves and the chromosomes condense and arrange on the spindle apparatus within the cytoplasm itself. Mitotic arrestants prevent formation of the spindle leaving the chromosomes free within the cell. Hypotonic solutions allow excess water to enter the cell, and this causes the chromosomes to spread out within the cytoplasm of the cell.
The degree of chromosome spreading is dependent on:
The composition of the hypotonic
The degree of hypotonicity
The length of hypotonic exposure time
The temperature of the solution
Common hypotonic solutions include:
0.075M, 0.06M, or 0.4% Potassium chloride (KCl)
Dilute medium
0.4% Sodium citrate
Water
3:1 KCL:NaCitrate

Question 56

Fixation Process

Answer 56

3:1 Methanol:glacial acetic acid is used to fix cells for air-dry chromosome preparations. This mixture is also known as Carnoy’s fixative.
Carnoy’s fixative kills the cells, and fixes the cell membrane. It precipitates proteins and removes the H1 fraction of the histone proteins from the chromosomes.
Acid-methanol fixation is a prerequisite for chromosome banding procedures. In peripheral blood harvests it also precipitates the free hemoglobin spilled by the red blood cells after lysis by the prior hypotonic treatment.
Cells with fixed membranes are brittle, allowing the cell membrane to break and disperse during the slide dropping procedure. Cells should be fixed until the cell pellet appears diffused and light. Washing cells with fixative more than 3 times, or using a higher concentration of acetic acid to methanol can result in a phenomenon sometime called “overfixing”. This is when the proteins are denatured to the extent that the cell membrane is brittle enough to break as soon as the cells are dropped on the slide or even before the cells are dropped onto the slide. This can result in overspreading of chromosomes. If “overfixation” occurs, the number of intact metaphases per slide will be reduced and a higher proportion of cells will have missing chromosomes. “Overfixation” will also cause the bonds which hold the sister chromatids together to break easier. Sister chromatids, which are widely spread, will give a lower quality of G-banding as compared to sister chromatids that are close together.

Question 57

Cell Pellet Storage

Answer 57

Leftover cell suspension may be stored at –20 o C for 2 to five years in 3:1 methanol:acetic acid fixative. It is important to store these fixed cells in an airtight container (either glass, polypropylene or polystyrene) to prevent evaporation.

Whenever you want to drop more slides, the cell suspension should first be centrifuged. The old fixative should be discarded, and the cells should be resuspended in fresh fixative.

Cells in fixative cannot be stored for more than overnight at 4 o C. At this temperature, the acid and methanol react to form an ester. Esters are sweet smelling. This conversion of the fixative retards cell spreading because of altered evaporation properties. When your fixative is sweet smelling, the cells should be centrifuged and resuspended in fresh fixative

Question 58

slides preparation

Answer 58

Slides should be composed of high quality crown glass with a refractive index of 1.515. They should be precleaned and free of the grease, which is used during the glass cutting procedure. Suitable clean slides when dipped in a container of water should retain rather than repel the water.

To enhance spreading, fixed cells should be dropped on wet slides. The Carnoy’s fixative will repel the water and this force can aid in the spreading of chromosomes. In addition, the cell suspension should be dropped at a 45o angle from top to bottom. This will also improve the spreading of chromosomes.

Question 59

slides quality

Answer 59

Optimal quality slides should have neither too many nor too few cells per field of view. Too few cells increase the amount of time needed to find the required number of metaphases for analysis. However, too many cells inhibit chromosome spreading, decreasing the likelihood of acquiring good crisp bands during the G-band procedure.
The presence of some cytoplasm is necessary to prevent the scattering of chromosomes across the slide. However, a visual cell membrane and poorly spread chromosomes encased in a large amount of cytoplasm will cause a reduction in the quality of banding. Properly treated slides should have metaphases with well-spread chromosomes in a circular formation. Only a light cytoplasmic background should be observed under a phase microscope and the chromosomes should have a medium gray appearance (Donlon’s Category II and III cells give the best G-banding results). A shiny appearance would indicate that the chromosomes are underfixed and spreading of the sister chromatids would indicate that the chromosomes are overfixed.

A phase microscope is a useful tool in the determination of cell density and quality. The most effective troubleshooting takes place during the slide dropping procedure.

Question 60

troubleshooting

Answer 60

Ambient conditions (e.g., temperature, humidity, air velocity)

Temperature - is one of the three important factors which influences the quality of chromosome preparations. This is because temperature directly affects the process of evaporation. A high temperature increases the rate of evaporation and a low temperature decreases the rate of evaporation. Both extremes will reduce chromosome spreading when humidity levels are optimal.
Humidity - has the same effect on chromosome preparations as temperature. When the humidity is too low slides dry too quickly and when humidity is too high, slides dry too slow.
If slides are drying too fast, more methanol can be added to the fix, slides can be dried near a humidifier or drying chamber, or cold slides may be used.
If slides are drying too slowly, warm slides may be used to increase evaporation rate or the airflow around the slides may be increased.
Air velocity - Slides processed in areas where there is a breeze (spinning centrifuges, air vents, and drafty areas) often have poorly spread or uneven appearing chromosomes. This is due to the slide either drying too quickly or unevenly.
A proper balance of temperature and humidity will result in good chromosome spreading and cytoplasmic dispersal. The evaporation process induced by the presence of methanol in the fixative provides a significant force, which can enhance chromosome spreading.
The temperature and humidity range for optimal chromosome preparations will vary somewhat from one geographical location to another. One report suggests the relative humidity of 50 to 60% combined with an ambient temperature of 24o to 27o C as being an optimal environment for slide preparation (Verma and Babu 1989).
NOTE: Proper hypotonic treatment is also a prerequisite of good chromosome spreading; yet without the proper ambient conditions, optimal chromosome preparations can not be obtained.

Question 61

troubleshoot (Chromosome scattering)

Answer 61

1. Can occur if the hypotonic solution has a lower hypotonicity than is recommended. Overspreading can also result from errors made in the preparation of the hypotonic solution, leaving cells too long in hypotonic, or from mixing the cells too hard in hypotonic.
2. Overfixation - Too many washes in fixative can result in overspreading of the chromosomes. After three washes, a test slide should be made to check for slide quality. Only if chromosomes are underspreading should more washes be performed. Cells should also be mixed gently once they are in fixative in order to prevent damage to the cell membrane that becomes hard and brittle with exposure to the fixative solution.

Question 62

troubleshoot(Chromosomes encased in cytoplasm)

Answer 62

1. This can occur if the volume of hypotonic is insufficient. If in doubt about whether enough hypotonic has been added to cells, extra hypotonic should be added. Too little hypotonic is more detrimental to the harvesting procedure than too much hypotonic. In other words, the more cells you have the more hypotonic solution would be added. Also, if the hypotonicity of the solution or the time of exposure is inadequate, the same result will be obtained. Excessive cytoplasm around the chromosomes will hinder banding efforts considerably.
2. Improper temperature, humidity or air velocity as discussed earlier will result in underspread chromosomes. Slides dipped in cold water can help improve spreading when the humidity is below the optimal level. This helps slow the evaporation process allowing the chromosomes sufficient time to spread. Conversely, slides dipped in warm water can help improve spreading when the humidity is above the optimal range by enhancing evaporation allowing the force of the evaporation process to help spread the chromosomes.
3. Underfixation of cells can have the same result as an insufficient hypotonic exposure. The cell membrane, if underfixed, will not break during dropping of the cells on the slide or during exposure to the forces of evaporation. If this occurs, some cytogeneticists will suggest dropping the cells from a greater height or with a greater force, hoping that slapping the cells against the slide will break the cell membrane.

Question 63

troubleshoot (Contracted chromosomes)

Answer 63

will result if the exposure to a mitotic arrestant is too long. Metaphase chromosomes will naturally contract during the metaphase period of cell division. However, if chromosomes are held in this state in the presence of a mitotic arrestant, the chromosomes can supercondense beyond the natural contraction state. Mitotic arrestants have been shown to bind to the chromosome and have an effect on chromosome contraction that is concentration and time dependent.

Question 64

The Effect of the Environment on Slide Making

Answer 64

1. hot&wet (evaporation too slow => overspreading): drop on dry slides, try warm slides, wave slides, increase(methanol)
2. hot&dry (evaporation too fast => underspreading): drop on wet slides, dry slides on wet paper towel or near humidifier
3. cold&wet (evaporation too slow)
4. cold&dry (evaporation too fast)

Question 65

Harvest Errors Resulting from Improper Reagent Sequence and Outdated Reagents

Answer 65

If reagents are used in an improper sequence, it can result in a poor quality or failed harvest. For example, if fixative is added to the specimen accidentally before the hypotonic treatment step, cellular proteins are fixed and nucleic acids are precipitated. This kills the cells. Cell death shuts down the process of osmosis, which is necessary for cell swelling. This is the primary requirement for chromosome spreading. Since this is a nonreversible error that will result in a harvest failure, it is wise to not have both the fixative and hypotonic solutions together when harvesting.
If hypotonic solution is added by mistake instead of fixative during the fixative washes, immediately add fixative to the hypotonic. Centrifuge the specimen for 10 minutes, discard the hypotonic and replace with fixative and complete the wash sequence. This error will not result in a failed harvest, however it may result in poor quality.

Outdated reagents

To avoid potential failure, it is important to monitor the reagents used. It is important to keep track of the catalog number, lot number, date opened, date received and most importantly the expiration date of your solutions. This includes the mitotic arrestants, hypotonic, methanol, acetic acid, intercalation agents and cell synchronization agents used during this process.
Each new batch of hypotonic solution should be evaluated by comparing new hypotonic solutions to that already in use to ensure successful harvest.