Cells in Culture

Part 1: Terminology

Cell Culture
Pros Use of animals reduced Cells from one cell line are homogenous and have same growth requirements, optimizing growing patterns. In vitro models allow for control of the extracellular environment Able to monitor various elements and secretions without interference from other biological molecules that occurs in vivo
The maintenance of cells outside of the living animal (in vitro) for easier experimental manipulation and regulation of controls.

 Cons Removal of cells from their in vivo environment means removing the cells, hormones, support structures and various other chemicals that the cells interact with in vivo. It is nearly impossible to recreate the in vivo environment. The artificial conditions could cause cells to de-differentiate which will cause them to behave differently and produce proteins other than it would in vivo.
Genotype: the genetic make-up of the cell Phenotype: the appearance and behavior of a cell as a result of their genotype. Most often, scientists are looking at phenotypic changes in their analysis of cells in culture

Classification of Cell Cultures

Primary Culture Cells taken directly from a tissue to a dish Secondary Culture Cells taken from a primary culture and passed or divided in vitro. These cells have a limited number of divisions or passages. After the limit, they will undergo apoptosis.
Apoptosis is programmed cell death

Making a Primary Culture

Cell Lines
Cell Line Cells that have undergone a mutation and wont undergo apoptosis after a limited number of passages. They will grow indefinitely. Transformed cell line A cell line that has been transformed by a tumor inducing virus or chemical. Can cause tumors if injected into animal. Hybrid cell line (hybridoma) Two cell types fused together with characteristics of each

Growing Cells in Culture

Part 2: Understanding Cell Behavior

Confluency
How covered the growing surface appears This is usually a guess Optimal confluency for moving cells to a new dish is 70-80%
too low, cells will be in lag phase and wont proliferate Too high and cells may undergo unfavorable changes and will be difficult to remove from plate.

Contact Inhibition
When cells contact each other, they cease their growth. Cells arrest in G0 phase of the cell cycle Transformed cells will continue to proliferate and pile upon each other

Anchorage Dependence
Cells that attach to surfaces in vivo require a surface to attach to in vitro.
Other cells or specially treated plastic or other biologically active coatings

Blood cells are primary exception. Transformed cells may not require attachment.

Passage number
The number of times the cells have been removed (or split) from the plate and replated.
Always write this on your plate or flask as P#

Growing Cells in Culture

Part 3: Solutions used in cell culture

Phosphate Buffered Saline - Ca2+ Mg2+ Free (PBS)

Used to wash/remove excess serum that inhibits the function of TrypsinEDTA. Must be warmed in the water bath before use so cells are not shocked by cold liquid.

Trypsin EDTA
An enzyme used to detach the cells from a culture dish. Trypsin cleaves peptide bonds (LYS or ARG) in fibronectin of the extracellular matrix. EDTA chelates calcium ions in the media that would normally inhibit trypsin.
Trypsin will self digest and become ineffective if left in water bath more than 20 minutes. Trypsinizing cells too long will reduce cell viability

Trypan Blue
An exclusion dye Living cells cannot take up the dye and will appear bright and refractile. Dead cells with broken membranes will absorb the dye and appear blue. Usually add 200 ml of trypan blue to 200 ml of cell suspension in eppendorf tube

Bleach
Used to destroy any remaining cells in dishes and tubes before they are tossed in the trash can. Add enough to change media to clear,
wait 5 minutes, rinse solution down sink throw away the dish/flask/plate in the trash can.

Growing Cells in Culture

Part 4 : Equipment

CO2 incubator

maintains CO2 level (5-10%), humidity and temperature (37o C) to simulate in vivo conditions.

Biosafety Cabinet

 Follow these start-up procedures when preparing for work : Turn off UV lights if in use and ensure that the sash is in the appropriate position. Turn on fluorescent light and cabinet blower, if off. Check the air intake and exhaust grilles for obstructions. If the cabinet is equipped with an alarm, test the alarm and switch it to the "on" position. Confirm inward airflow by holding a tissue at the middle of the edge of the viewing panel and ensuring that it is drawn in. Disinfect the interior surfaces with a suitable, noncorrosive disinfectant. Assemble all materials required for the procedure and load them into the cabinet; do not obstruct the air grilles; the working surface may be lined with absorbent paper with plastic backing; segregate "clean" items from "contaminated" items. Wait 5 minutes to purge airborne contaminants from the work area.

Follow these procedures for working in the cabinet : Wear protective clothing and gloves as appropriate. Perform operations as far to the rear of the work area as possible. Avoid movement of materials or excessive movement of hands and arms through the front access opening during use; when you do enter or exit the cabinet, do so from straight on; allow the cabinet to stabilize before resuming work. Keep discarded, contaminated material to the rear of the cabinet; do not discard materials in containers outside of the cabinet. Do not work with open flames inside the cabinet. If there is a spill during use, surface decontaminate all objects in the cabinet; disinfect the working area of the cabinet while it is still in operation (do not turn the cabinet off).

Follow these procedures upon completion of the work : Allow the cabinet to run for 5 minutes with no activity. Close or cover open containers before removing them from the cabinet. Surface disinfect objects in contact with contaminated material before removal from the cabinet. Remove contaminated gloves and dispose of them as appropriate; wash hands. Don clean gloves, and ensure that all materials are placed into biohazard bags within the cabinet. Using a suitable non-corrosive disinfectant (e.g., 70% ethanol), disinfect interior surfaces of cabinet Turn off the fluorescent light and cabinet blower when Turn on the UV light if appropriate (do not turn on when people are working close by

Water bath
To warm media, and PBS before placing on cells Can harbor fungi and bacteria, spray all items with 70% ethanol before placing in the hood. Usually takes 10 -15 minutes for media to warm, 5-10 for TRED to thaw

Vacuum pump
For permanent aspiration of liquids (media, PBS and TRED). Use unplugged glass pasteur pipets, throw into sharps box when done.

Inverted Phase Microscope

A phase contrast microscope with objectives below the specimen. A phase plate with an annulus will aid in exploiting differences in refractive indices in different areas of the cells and surrounding areas, creating contrast

Mechanics of phase microscopy

Shifting of phase by a wavelength Add and subtract amplitudes to create more contrast

A comparison

Phase contrast microscopy Can be used on living cells

Light microscopy requires stain, thus killing cells

Basic cell culture instructions

Aseptic Technique
For best results in tissue culture, we want to work to keep microbial (bacteria, yeast and molds) contamination to a minimum. To do this, there are certain things you must be aware of and guidelines to follow. Work in a culture hood set-aside for tissue culture purposes. Most have filtered air that blows across the surface to keep microbes from settling in the hood. Turn off the UV/antimicrobial light and turn on the hood 30 minutes prior to entering the hood. Wear short sleeves or roll your sleeves up. Turn your baseball caps back if you MUST wear them, tie long hair back and remove rings and watches.

 Wash hands with soap and water before beginning the procedure and rewash if you touch anything that is not sterile or within the hood.
Spray down your hands, work surface, and anything that will go into the hood with 70% ethanol. Rewipe at intervals if you are working for a long time in the hood. This will reduce the numbers of bacteria and mold considerably. Do not breathe directly into your cultures, bottles of media, etc. This also means to keep talking to a minimum. No singing or chewing gum.

 Work as quickly as you can within limits of your coordination. Also, keep bottles and flasks closed when you are not working with them. Avoid passing your arm or hand over an open bottle.
Use only sterilized pipets, plates, flasks and bottles in the hood for procedures. Take special precautions with the sterile pipets. Remove them from the package just before use. Make certain to set up the numbers on the pipet so that they face you. Never mouth-pipet, use the pipetting aid. Change pipets for each manipulation. If the tip of the pipet touches something outside of the flask or bottle, replace with a new one. Never use a pipet twice.

Basic Cell Culture Procedure for Anchorage Dependent Cells View cells using inverted phase microscope Aseptically aspirate media Rinse media with PBS Add Trypsin-EDTA to cells Aspirate Trypsin-EDTA Incubate cells with layer of Trypsin-EDTA at 37 C Resuspend cells with fresh media Take sample and count cells Calculate how many cells are needed to add to new plate or flask

Some volumes dont need to be exact in cell culture

Rinsing volume of PBS (as long as it fits in the dish and is sufficient to rinse the serum). Volume of trypsin EDTA as long a bottom of plate or flask can be covered. Volume of media used to resuspend your cells. The same number of cells will be there despite the volume of media used. Too little resuspension media will result in very high cell count and would require more dilution (and higher dilution factor). The volume needed to seed your next plate would then be very small, maybe too small to work with. Too much media would result in low cell count/ml and you may need a large volume to add to your newplate.

 Volume of cells removed for cell counting.

You want enough to work with, but not take all of your cells from your plate. If you want a dilution factor of 2, just add an equal amount of trypan blue.

Exact # of cells to be plated

If you want to plate 2 x 10 5 cells onto your plate, but you have 2.1 x 10 5 cells/ml, plating 1ml will be easier than plating .953 ml.

Troubleshooting Low Hemacytometer Counts

Trypsinization not complete

Trypsin is ineffective
too cold, be sure to warm sufficiently self digested or expired check date, don't warm too long too much serum left on plate rinse plate thoroughly with PBS

Trypsinization technique
Trypsin doesn't coat plate, completely add full 2 mls, lay flask down, count to 10, then remove trypsin left on plate too long and then aspirated...cells removed along with trypsin not left long enough in incubator depends on cell line 3T3-L1 can go 1-5 minutes flask may need to be tapped or slapped to facilitate cell removal (this varies by cell line, but ok for 3T3s)

Resuspension technique
too much media added more media results in low cell/ml, but overall cells on plate should remain the same cells not sprayed off surface properly media and cells not pipetted (gently) up and down 3-4 times to break up clumps too long of time before retrieving sample from flask (cells may settle). After mixing with trypan, don't wait too long before loading hemacytometer. Get hemacytometer ready while trypsinizing cells in incubator

Stubborn cells
cells left on plate a long time (>4 days) will be more difficult to remove very confluent plate will require more aggressive trypsinization because trypsin cannot recach plate surface effectively

Keeping a good lab notebook

 Lab notebooks provide a convenient place for you to keep all of your procedures, data and observations in one place. If written well, a lab notebook should contain everything you need to know to allow you or someone else to repeat any experiment you have ever performed. It can be useful in finding the source of errors and unexpected results when problems arise. Should your work ever be disputed, a lab notebook will provide testimony to your research. By following the simple guidelines below, you will learn how to keep a good lab notebook.

The notebook should be bound (no spiral notebooks, please). The pages should be numbered either by hand or preprinted before using the book. Use only permanent ink. Write your name, contact information, and dates the notebook covers on the first page. Skip the next 2-3 pages for a Table of Contents. Fill in the experiment name and page numbers as they are completed. Write the date, experiment title, and partners name at the top of each page.

The first time you use a procedure

Write the whole procedure in your own words into the notebook OR tape in the typed version Include a reference to the lab manual page or the published procedure. Note any changes made to the original procedure. Do not just copy the lab manual or procedure word for word; restate each step simply and clearly. If you repeat this procedure later, reference the page where it was first performed and write down any changes made.

 All data and observations should be written in your notebook at the time you took the measurement. Do not write on scratch paper to be copied later into your notebook little pieces of paper may be lost and data forever lost.
Remember your lab notebook is extemporaneous writing. Keep it neat but do not waste too much time making it perfect. Errors should be crossed out with a single line (example). Do not scribble out mistakes.

 Write down all calculations, no matter how simple, in your notebook. For example, every time you perform a cell count, cell viability must be calculated and recorded. Permanently attach (glue or tape) images, computer print outs, and other data in your notebook. Date and initial over the corner of the attachment. Be sure to label the image with any pertinent information. [For example, if you place a Western Blot image into your notebook, label the lanes with what was in each, and the gel composition. If the lysates were prepared on a date different from the date the gel was run make a reference to the page that contains information on how the lysates were made.] Partners may photocopy original data for inclusion in the lab notebook.

 Including complete chemical equations, statistical equations, sample calculations, and sketches or block diagrams of any apparatus used is also good practice. Record start and stop times. Include conclusions from this data. What does it mean and did it work as expected? If unexpected results occur, explain why. Include expected values (with reference) where appropriate. Do not skip pages. Use every page of the notebook. If you need to rewrite a page, draw a large X through the page, date, initial, and start over on the next page. The same applies if you dont fill an entire page draw a line through the remaining space, date, and initial.

Six Essential Calculations

Hemacytometer
Specialized chamber with etched grid used to count the number of cells in a sample. use of trypan blue allows differentiation between living and dead cells

Using the Hemacytometer

Remove the hemacytometer and coverslip (carefully) from EtOH and dry thoroughly with a kimwipe.

Center coverslip on hemacytometer

Barely fill the grid under the coverslip via the divet with your cell suspension. Count cells in ten squares (5 on each side) by following diagram at station.

Looking at the grid under the phase contrast microscope

How the cells will appear

Bright refractile spheres are living cells, Blue cells about the same size as the other cells are dead.

Keep a differential count of blue vs. clear for viability determination. Sometimes there will be serum debris, and this will look red or blue and stringy or gloppy-dont count it!

These are blood cells, You will not have this many

Count 10 squares Any 10 will do but we will follow convention Watch for stringy, reddish materialthose arent cells!
serum

Top group
Count cells that touch top and left lines

DO NOT
Count cells that touch bottom and right lines

Bottom Group

Calculate your cells/ml

Calculate the number of total cells in one ml of your suspension.
Total cells counted x (dilution factor) x (10,000) number of squares

Here, dilution factor is 2 and # of squares is 10 (our example 62/10 x2 x104 =1.24 x 105)

Determine your percent viability

Viability is a measure how many of your cells survived your cell culture technique.
# of viable (living) cells x 100 total number of cells counted Our example 54/62 x 100 =87.09%

Calculate total # of cells in original suspension

Number of cells per ml x total mls of original suspension Lets assume 10ml original suspension 1.24 x105 x 10 =1.24 x 106 cell total

Total # of viable cells available in original suspension

Total number of cells in original suspension x % viability 1.24x106 x 87% =1.08x 106 viable cells in the original suspension

Determine the number of cells you need to add to your flask

You want the cells to grow happily without overcrowding (or being too sparse) before the next time you come into class. Using the calculation on the next slide, figure out the number of cells needed for the size of vessel being used You need to take into account:
length of time cells are to be grown. the size of the cells (not directly in the formula) their doubling time

Determine how many mls of cell suspension much to add to your flask
# of cells needed cells/ml

Growing Cells in Culture

Part 5: The protocol

Observing cells in culture

Check color of media
Healthy growth usually leaves media slightly orange Too yellow means bacterial growth Too purple means low carbon dioxide, cells dead

Observe cells under phase microscope

Spread out or rounded? How confluent?

What to do with growing cells

If they are at least 7080% confluent Subculture them
Also called passing or splitting

If they are not very confluent Lift and replace onto same plate
Culture more than 4 days old for our cells Remove old media, lift cells from plate and resuspend in fresh media on same plate

Remove media, remove cells, resuspend and transfer some to a new plate

Feed them
Culture less than 4 days old Remove old media and replace with fresh, warm

Brief subculturing preview

Remove media, lift cells from plate Resuspend cells in fresh media Count cells and determine viability Seed new plates with appropriate # of cells and volume of media

You will need to return to take care of your cells

Thursday or Friday is an in between point before next week. First time through may require up to an hour If one member cannot make the return time, that person should work in hood tonight. Choose times that will be consistent each week