lecture4- monolayer cell cultures (1)

© 2009, Maulik Suthar

Subculturing Monolayer Cell Cultures

Maulik P. Suthar


Introduction

• Most animal cell lines and primary cultures grow as a single thickness cell layer or sheet attached to a plastic or glass substrate.

• Once the available substrate surface is covered by cells (a confluent culture), growth slows and then ceases.

• Why subculture? : to keep the cells healthy and actively growing • Subcultivation process involves breaking the bonds or cellular ‘glue’ that

attaches the cells to the substrate and to each other by using proteolytic enzymes such as trypsin, dispase, or collagenase.

• Combined with divalent cation chelators such as EDTA (binds calcium and magnesium ions).

• The loosened cells are then removed from the culture vessel,counted, diluted and subdivided into new vessels.

• Cells then reattach, begin to grow and divide, and, after a suitable incubation period (depending on the initial inoculum size, growth conditions and cell line), again reach saturation or confluency. At this point, the subcultivation cycle can be repeated.


Sterile requirements

1. Flask of actively growing cells that are 80 to 90% confluent

2. Culture medium. This should contain all of the additives (fetal bovine serum, glutamine etc.) required by the above cell line.

3. Phosphate-Buffered Saline (Free from Calcium- and Magnesium)-- is used to maintain proper pH and osmotic balance while the cells are being washed to remove protease inhibitors that are found in most animal sera.

4. 0.1% Trypsin solution: Trypsin is normally used in concentrations ranging from 0.05% to 0.25%. Working concentrations are usually determined by using the lowest trypsin concentration that can remove the cells from the substrate and give a single cell suspension in a relatively short time (5 to 10 minutes). Trypsin solutions are often supplemented with other enzymes (collagenase) or chelating agents (EDTA) to improve its performance.

5. Centrifuge tubes - 15mL disposable screw cap

6. Culture vessels : Appropriate type and size

7. Pipettes -1, 5, 10 and 25mL -

8. 0.04% Trypan blue (Sterile) solution for viablity staining

9. Pipette tips

10.Laminar flow hood


Procedure

1. Examination:examine cultures daily and always prior to subcultivation. Using an inverted phase contrast microscope (100 to 200x), quickly check

the general appearance of culture. Look for signs of microbial contamination. Many cells round up during mitosis, forming very refractile (bright) spheres

that may float free of the surface when the culture is disturbed. Dead cells often round up and become detached but are usually not bright or

refractile.

2. Cell harvesting:This step removes the cells from the plastic substrate and breaks cell-to-cell

bonds as gently as possible. When using enzymatic dissociation: a) the old medium is removed and discarded; b) the cell monolayer is gently rinsed; c) the enzyme solution is added and the culture incubated until the cells

are released.


Procedure

c) Add 5mL of the trypsin solution (in CMF-PBS) to the flask and place the flask back in an incubator at 37ºC to increase the activity of the enzyme solution. (Prewarming of the enzyme solution to 37ºC will decrease the required exposure period.)

d) Check the progress of the enzyme treatment every few minutes with an inverted phase contrast microscope. Once most of the cells have rounded up, gently tap the side of the flask to detach them from the plastic surface. Then add 5mL of growth medium to the cell suspension and, using a 10mL pipette, vigorously wash any remaining cells from the bottom of the culture vessel. At this point a quick check on the inverted microscope should show that the cell suspension consists of at least 95% single cells. If this is not the case, more vigorous pipetting may be necessary

e) Collect the suspended cells in a 15mL centrifuge tube and place on ice. Some dissociating agents should be removed at this point by centrifugation to prevent carry over which can cause poor cell attachment or toxicity. However, the trypsin in the cell suspension will be inactivated by the serum and does not absolutely need to be removed. If removal is desired, spin the cell suspension at 100xg for 5 minutes. Then remove the trypsincontaining medium and replace with fresh medium.


To determine growth rates or set up cultures at known concentrations it is necessary to count the cell suspension. Hemacytometers or electronic cell counting devices can be used.

The hemacytometer has the added advantages of both being less expensive and allowing cell viability determinations to be made during counting.

Vortex the cell suspension and remove a 0.5mL sample and place in a tube for counting. To this add 1mL of the vital stain trypan blue (0.04%).

Mix well by vortexing, withdraw a 20μL sample with a wide tip pipettor and carefully load a clean hemacytometer. (Do not overfill!)

Do a viable cell count and calculate the number of viable cells/mL and the total cell number.

Cell counting:


• After making the appropriate dilutions, add the correct amount of cells to each culture vessel. Then add fresh medium to bring the culture vessel to its recommended working volume.

• label all vessels accurately; write on the sides of flasks and around the outer edge of the dish tops so as not to interfere with microscopic observation.

Plating: How much and how many?


Most mammalian cell cultures – optimum temperature : between 35º and 37ºC.

High humidity levels and CO2 concentrations. The high humidity cuts down evaporation losses in open systems such as petri dishes and microplates that would otherwise result in hypertonic culture medium and stressed cells. The elevated CO2 concentrations (usually 5% to 10%, depending on bicarbonate concentrations in the medium) help maintain the proper pH (7.4 ± 0.2) when used with the correct bicarbonate buffer system.

In order for this type of buffer system to work it is necessary to allow gas exchange by using unsealed dishes and plates or flasks with gas permeable (vented) caps.

Leave caps on flasks slightly loosened (or use vented caps on the flasks for extra protection against spillage and contamination) and place on a shelf in a 37ºC, humidified CO2 incubator.

Examine cultures daily and change medium as needed.

Incubation


Trypsinization Procedure


Requirements

1. Culture medium: all of the additives (sera, glutamine, etc.) required by the chosen cell line. 2. Calcium- and Magnesium-Free Phosphate-Buffered Saline -PBS (10mL). This simple salt solution is used to maintain proper pH and osmotic balance while the cells are being washed to remove protease inhibitors that are found in FBS.

2. 0.05% Trypsin/EDTA (0.05%) solution: Trypsin is normally used in concentrations ranging from 0.05% to 0.25%. Working concentrations are usually determined by using the lowest trypsin concentration that can remove the cells from the substrate and give a single cell suspension in a relatively short time (5 to 10 minutes). Trypsin solutions are often supplemented with other enzymes (collagenase) or chelating agents (EDTA) to improve its performance.

3. 15mL disposable screw cap centrifuge tubes 4. Hemacytometer5. Centrifuge6. Phase contrast microscope7. 1, 5, 10 and 25mL pipettes


Procedure1. Aseptically aspirate spent media from the bottom well of the plate 2. Rinse the top and bottom of the well insert 2 to 3 times with PBS to remove any serum (contains

trypsin inhibitors). Review chart on left for recommended buffer volumes.3. Add Trypsin/EDTA to the bottom of the well. Use the same liquid volumes as above. Incubate for

10 minutes in a 37°C incubator, containing 5% CO2.4. After 5 minutes, view the cells under a phase contrast microscope for detachment. Generally,

the lower the trypsin concentration and the less time the cells stay in trypsin the better. 5. Once the cells look rounded or detached (approximately 10 minutes), remove the Trypsin/EDTA

containing cell suspension and add to a 15mL centrifuge tube containing 5mL of serumcontaining culture medium to inactivate the trypsin. (For serum-free culture use a trypsin inhibitor.)

5. With the cover on the plate of the Transwell inserts, sharply hit the side of the plate with your hand to detach cells from the membrane. Rinse the top and bottom of the Transwell insert with Trypsin/EDTA to remove any remaining cells. Add the cell suspension to the centrifuge tube.

6. Observe Transwell inserts under a phase contrast microscope to ensure that all of your cells have been removed. Perform a second trypsinization if there are large numbers of cells still remaining on the membrane by repeating the steps 3 to 5.

7. Gently vortex the cells to resuspend and then remove an aliquot of cell suspension and count with a clean hemacytometer. Centrifuge cells for 5 to 10 minutes at 100xg. Remove media from the centrifuge tube, leaving the cell pellet, and resuspend cells in the appropriate amount of growth media.


Fixation and Staining


Requirements

• Reagents• Phosphate Buffered Saline (for cell culture)• 2.5% Glutaraldehyde, EM Grade 1.06 (Polysciences)• 0.5% Triton X-100• Gill's Hematoxylin No.1 (Polysciences) Filter through a vacuum filter with a• 0.2μm membrane.• Acid alcohol: 0.5% Hydrochloric Acid in 70% ethanol• 0.04%Ammonium Hydroxide• Materials• Fume hood• Pipette aid and pipettes• Modified cluster dishes (holes in the cover and base )• Small basin or container


Procedure

1. Rinse the Transwell membrane with cells by adding PBS (37°C) to the bottom of the well plate until it reaches the membrane, and then add PBS to the top of the membrane. The rinse should be added slowly and then aspirated. Do not touch either surface of the membrane. Repeat the rinse procedure.

2. Pipette enough PBS to cover surface of the membrane. PBS will remove any residual growth media. Remove the PBS from the Transwell insert.

3. Pipette enough 2.5% EM grade glutaraldehyde to cover the surface of the membrane. Leaving the glutaraldehyde in the Transwell insert, incubate at room temperature for 15 minutes. Remove glutaraldehyde by carefully aspirating it or pouring it off.

4. Pipette enough 0.5% Triton X-100 to cover the surface of the membrane. Leaving the Triton X-100 in the Transwell insert, incubate for three minutes at room temperature. Remove Triton X-100 from the Transwell insert in the manner described in step 3.


5. Pipette enough Gill's hematoxylin No.1 to cover the surface of the membrane. Leaving the hematoxylin in the Transwell insert, incubate at room temperature for 15 minutes. Remove the hematoxylin from the Transwell insert in the manner described in step 3.

6. Rinse Transwell insert 3 to 4 times in a basin filled with distilled water to remove the excess stain.

7. Pipette enough acid alcohol to cover the surface of the membrane and leave it for 2 to 3 minutes to remove any residual stain (destain).

8. Rinse Transwell insert twice in a basin filled with fresh distilled water.

9. Pipette enough 0.04% NH4OH to cover the surface of the membrane and leave it until a blue color is observed on the membrane (2-3 minutes).

10. Rinse Transwell insert twice in a basin filled with distilled water.

11. Air dry at an angle on a clean paper towel overnight.


Clonal Growth of Cells in Semisolid Media


Introduction

• Established cell lines such as HeLa and L-cells, as well as normal cells transformed by viruses, can form colonies suspended in soft agar-based media. Most normal cells will not grow under these conditions although there are exceptions (e.g. cartilage).

• The baby hamster kidney line, BHK-21, will not grow in agar, but will after transformation by polyoma virus (1, 2). In contrast, if agarose is used, (a purified agar, free of sulfated polysaccharides) BHK-21 will grow in suspension. If all these factors are taken into consideration and standardized, this system can be used to measure “transformation." It must be remembered, however, that morphological transformation and ability to grow in suspension are not necessarily correlated with the ability to form tumors in appropriate hosts.

• Growth of cells in semisolid medium, whether agar, agarose, or methylcellulose offers a second advantage. The bacteria like colonies that form from monodispersed cell suspensions offer a means of isolating clones with a minimal amount of effort.

• Using a finely drawn pipette, single, well isolated colonies can be removed from the suspended state and subcultured. However, there are variations that must be used depending upon cell types. Clones in which there is loose intercellular bonding can be dissociated into a monodispersed population through gentle pipetting. However, many cell types require further enzymatic treatment to disperse them or must be treated as explants.


Requirements

1. 20mL of 2.5% Bacto-Agar (Difco) in distilled water in 100mL glass bottle - Corning Cat. # 1395-100 or 1396-100. Solution should be sterilized by autoclaving. Agar needs to be melted at 100°C prior to use and kept at 45°C until mixed with Nutrient Mix.

2. 50mL 1X base medium - sterile. This should be made from the standard medium (no serum) used to grow the cells that will be cloned.

3. 20mL 2X base medium - sterile. Reconstituting 10X-powdered standardmedium with only half the required water (no serum) is used to make the 2X medium.

4. 50mL complete growth medium – sterile. (This should be made from base medium plus 10% fetal bovine serum) for dilutions. Base medium should be the same medium that is normally used to grow the cell culture.

5. Fetal bovine serum (10mL)6. 80mL Nutrient Mix in 100mL glass bottle - Corning Cat. # 1395-100 or 1396-100 Make up by

combining: 2X base medium (20mL)Fetal bovine serum (10mL)1X base medium (50mL)7. 60mm plastic dishes - Corning Cat. # 430166 (8)8. 15mL plastic centrifuge tubes - Corning Cat. # 430055 or 430789 (16)9. 10mL pipettes - Corning Cat. #4488 or 4101 (1 bag)10. 1mL pipettes - Corning Cat. #4485, 4011 or 4012 (1 bag)11. Water baths at 45°C and 100°C12. Cell suspension for plating (1mL at 106 cells/mL)


Procedure

Label and prepare all plates and dilution tubes in advance.

1. Prepare 1X nutrient agar medium:

a) Melt 2.5% agar in autoclave, microwave oven or boiling water bath, then place in 45°C water bath. The agar temperature must be allowed to cool to 45°C before proceeding.

b) Warm nutrient mix in 45°C water bath. The temperature of the nutrient mix must be allowed to reach 45°C before proceeding.

c) Pour contents of 2.5% agar into nutrient mix to create the osmotically balanced 1X nutrient agar medium with a 0.5% agar concentration. Mix gently but AVOID BUBBLES. Do not allow mixture to cool. Keep nutrient agar medium in the 45°C water bath when not being used.

2. Pipette 7mL of the 1X nutrient agar medium per 60mm dish. Allow agar to cool and harden. Once hardened, return the plates to the incubator. This agar layer will provide a base nutrient layer to support cell growth for at least one week. It will also keep the cells from reaching and attaching to the plastic on the bottom of the dish.


3. Distribute 1mL aliquots of 1X nutrient agar medium in eight 15mL centrifuge tubes. Keep tubes at 45°C in water bath and do not allow mixture to cool or it will begin to harden and develop clumps.

4. Prepare the cell suspension in complete growth medium. When first plating a new cell type, we recommend that tubes be set up with the following cell concentrations: 1x105, 1x104, 1x103, and 1x102 cells/mL. Use 0.5mL cell suspension added to 4.5mL complete growth medium (no agar) to make these 1:10 dilutions.

a) Add 0.5mL of each dilution to individual tubes of the nutrient agar mixture. Mix gently (but avoid bubbles) and immediately pour contents of the tube (0.33% agar) on top of the bottom agar layer in one of the dishes. Work rapidly. If the nutrient agar is lower than 45°C prior to mixing, then the cell suspension may form clumps when plated. If the medium is too warm, the cells will be heat-shocked and may not survive.

b) Repeat the process for the seven remaining tubes and plates, setting up each cell concentration in duplicate.

5. Allow agar in plates to harden for 15 to 30 minutes on the bench top and then place them in a CO2 incubator. If the resulting medium is too soft, try increasing the initial agar concentration to 3.5%. This will give a final agar concentration in the base layer of 0.7%.

6. Examine plates every two or three days until colonies are large enough to see with the unaided eye.


Suspension Culture Issues

• Reduce Shearing Damage Because animal cells lack a protective cell wall, they can be easily damaged by the shear forces that develop if they are spun too fast or shaken too vigorously. Some cell lines, especially those grown in serum-free media, are very sensitive to shear forces and a difference of only 30 to 50 RPM in mixing speeds can lead to serious growth problems. For many cell lines, shaking them is often gentler than stirring them and is a better approach when trying to suspension-adapt cells.

• For shaker flasks, it is recommended to start with a shaking rate of 75 to 150 RPM on an orbital shaker with a medium volume of 30 to 40% of the nominal flask capacity (1L of medium in a 3L flask). For Corning spinner flasks a starting speed of 50 to 150 RPM is suggested.

• The use of baffled spinner and shake flasks can reduce the required mixing rates considerably. Some cell types, such as insect cells, require higher oxygen levels and may benefit from more vigorous mixing and the use of vented flasks or continuous gassing. It is strongly recommended to empirically determine the optimum stirring or mixing conditions.

• Start by choosing the lowest speed that appears to give an even cell distribution from the top to the bottom of the flask. However, to get adequate gassing, higher speeds may be necessary, Shearing damage from these higher speeds can be reduced by increasing the medium viscosity by adding carboxymethylcellulose (1 to 2%), BSA (100 μg/mL) or Pluronic® F-68 (0.1%) to the medium (Mather, 1998b). This is especially important when using reduced serum or serum-free medium.


• Avoid Cell Clumping and Sticking: Some cell lines tend to form large clumps when grown in suspension. These larger clumps tend to settle to the bottom of the flask or may attach to the flask side walls and can result in lower cell viability and growth. Using a calcium-free medium (Joklik’s MEM; S-MEM) will reduce cell clumping as these divalent cations are very important in cell to cell binding (McLimans; 1979). Coating the surface of glass suspension flasks with siliconizing solutions before sterilization will reduce cell clumps forming on the flask surface. Sigmacote®, AquaSil™, and Siliclad® are some commercially available siliconizing solutions suitable for cell culture applications. It is important to carefully follow the use and safety directions for these products to avoid culture toxicity.

• Use Appropriate Seeding Densities: Using the correct initial seeding density is very important when growing cells in suspension. It is always better to add too many cells rather than too few. Start with a seeding density of 1 x 105 to 5 x 105 cells/mL; the higher concentration is better when cells are adapting to serum-free conditions. An alternative approach for spinner and shake flasks is to start with half the normal volume of medium. This reduces the number of cells required to reach the optimum seeding densities by 50%. After the cells are actively growing (after 24 to 48 hours), additional medium can be added to bring the vessel to its final operating volume.

• Avoid Overheating Cultures: Flask cultures placed directly over magnetic stirrers or shaker motors may overheat as the result of excessive heat transfer from the motors to the flasks. In walkin warm rooms this may affect only flasks placed directly over the motor but in small incubators it may cause the entire incubator to overheat. Check for this problem in advance by placing identical culture flasks filled with water in the incubator and monitoring the temperature for at least 48 hours. Sometimes heat transfer from the stirrer to the flask can be reduced by elevating the flask a few millimeters above the stirrer surface so that air can flow beneath it. Also make sure that any stirrers or shakers used in humidified CO2 incubators are designed to withstand the corrosive atmosphere. Placing shakers in incubators will usually generate vibrations which may prevent cells from attaching to culture vessels in the same or adjoining incubator chambers.


Attachment-Dependent Culture Issues

• Use Appropriate Seeding Densities: Starting with the correct initial seeding density is also very important when growing attachment-dependent cells. It is always better to add too many cells than too few; seeding densities can always be lowered later. Start with a seeding density of 104 to 2 x 104 cells/cm2; the higher concentration is better when using difficult to grow cells or cells are adapting to serum-free conditions

• Help Cells Attach Quickly: Cell attachment problems are often a serious problem, especially when growing cells in reduced- or serum-free medium. Prewarming the medium used for the initial cell seeding and pregassing larger culture vessels so the medium will reach its correct pH sooner will help cells attach more quickly. Pregassing larger vessels before seeding is highly recommended and should be done with filtered medical grade 5% CO2/95% air mixtures. For cells that have attachment problems on traditional cell culture vessel surfaces, Corning recommends trying the patented Corning® CellBIND® surface on flasks, roller bottles and CellSTACK® chambers This surface is created by a novel microwave plasma process that improves cell attachment by incorporating significantly more oxygen into the cell culture surface than traditional plasma or corona discharge treatments, rendering it more hydrophilic (wettable) and increasing the stability of the surface.


• Rotate Roller Bottles Slowly: The constant movement of the medium across the surface of the bottle, slow though it appears, can make it more difficult for cells to attach and grow in roller bottles compared to stationary vessels such as flasks and dishes (Figure 17). A recommended starting speed for initiating roller bottle cultures is 0.5 to 1.0 revolutions per minute (rpm) to start. However, if cells have difficulty attaching (or staying attached), slower speeds (0.1 to 0.4 RPM) should be used until the cells are attached (Clark et al.; 1990). The constant motion of the medium can also lead to a more stressful cell environment than is found in stationary culture systems. Consequently, any techniquerelated issues that reduce the attachment ability of cells are magnified and clearly stand out. Using prewarmed medium and pregassing the bottles with CO2 so that pH shifts are minimized when inoculating cells will make it easier for the cells to quickly attach.

• Keep the Cells Happy: Maintaining optimal cell to medium ratios is important for obtaining good cell growth. As a starting point, use 0.2 to 0.3 mL medium for each square centimeter of culture vessel growth surface area (i.e., 5 to 7.5 mL for a 25 cm2 flask). Using more medium may reduce the need for feeding (changing the medium) in the cultures but, due to the increased medium depth and the static nature of the environment, will also slow the diffusion of oxygen to the cells. (See also Feed cultures appropriately, page 11.) Sometimes gassing the cultures will increase cell yields and viability. This is not practical on dishes, flasks and roller bottles because of the large number of vessels involved but is useful with CellSTACK chambers. It is also important to subculture cells before they reach confluency to keep them actively growing and healthy. In addition, epithelial-like cells will often form very strong cell-cell bonds at confluency making them much harder to remove from the substrate when subculturing them.

• Harvest Cells Gently: Don’t over dissociate cells when subculturing or harvesting. Too long exposure to harsh dissociating agents can reduce viability and make it very difficult for cells to reattach. This often occurs when attempting to harvest too many vessels simultaneously. It is better to harvest vessels a few at a time rather than attempt to harvest many at once. If using serumfree medium, make sure the dissociating agents are either inactivated or removed by gentle centrifugation. Keep harvested cells chilled until ready to reseed new vessels. This will maintain viability and reduce cell clumping. There are a variety of dissociating enzymes and agents available; experimenting with different combinations may improve both harvesting efficiency and cell viability (Freshney; 2000).


Cell Culture: Applications

• Cell culture has become one of the major tools used in cell and molecular biology. Some of the important areas where cell culture is currently playing a major role are briefly described below:

• Model Systems : Cell cultures provide a good model system for studying 1) basic cell biology and biochemistry, 2) the interactions between disease-causing agents and cells, 3) the effects of drugs on cells, 4) the process and triggers for aging, and 5) nutritional studies.

• Toxicity Testing: Cultured cells are widely used alone or in conjunction with animal tests to study the effects of new drugs, cosmetics and chemicals on survival and growth in a wide variety of cell types. Especially important are liver- and kidney-derived cell cultures.

• Cancer Research: Since both normal cells and cancer cells can be grown in culture, the basic differences between them can be closely studied. By the use of chemicals, viruses and radiation, to convert normal cultured cells to cancer causing cells. Thus, the mechanisms that cause the change can be studied. Cultured cancer cells also serve as a test system to determine suitable drugs and methods for selectively destroying types of cancer.

• Virology : One of the earliest and major uses of cell culture is the replication of viruses in cell cultures (in place of animals) for use in vaccine production. Cell cultures are also widely used in the clinical detection and isolation of viruses, as well as basic research into how they grow and infect organisms.


• Cell-Based Manufacturing: While cultured cells can be used to produce many important products, three areas are generating the most interest. The first is the large-scale production of viruses for use in vaccine production. These include vaccines for polio, rabies, chicken pox, hepatitis B and measles.

• Second, is the large-scale production of cells that have been genetically engineered to produce proteins that have medicinal or commercial value. These include monoclonal antibodies, insulin, hormones, etc.

• Third, is the use of cells as replacement tissues and organs. Artificial skin for use in treating burns and ulcers is the first commercially available product. However, testing is underway on artificial organs such as pancreas, liver and kidney. A potential supply of replacement cells and tissues may come out of work currently being done with both embryonic and adult stem cells. These are cells that have the potential to differentiate into a variety of different cell types. It is hoped that learning how to control the development of these cells may offer new treatment approaches for a wide variety of medical conditions.

• Genetic Counseling: Amniocentesis, a diagnostic technique that enables doctors to remove and culture fetal cells from pregnant women, has given doctors an important tool for the early diagnosis of fetal disorders. These cells can then be examined for abnormalities in their chromosomes and genes using karyotyping, chromosome painting and other molecular techniques.



• Genetic Engineering: The ability to transfect or reprogram cultured cells with new genetic material (DNA and genes) has provided a major tool to molecular biologists wishing to study the cellular effects of the expression of theses genes (new proteins). These techniques can also be used to produce these new proteins in large quantity in cultured cells for further study. Insect cells are widely used as miniature cells factories to express substantial quantities of proteins that they manufacture after being infected with genetically engineered baculoviruses.

• Gene Therapy: The ability to genetically engineer cells has also led to their use for gene therapy. Cells can be removed from a patient lacking a functional gene and the missing or damaged gene can then be replaced. The cells can be grown for a while in culture and then replaced into the patient. An alternative approach is to place the missing gene into a viral vector and then “infect’’ the patient with the virus in the hope that the missing gene will then be expressed in the patient’s cells.

• Drug Screening and Development: Cell-based assays have become increasingly important for the pharmaceutical industry, not just for cytotoxicity testing but also for high throughput screening of compounds that may have potential use as drugs. Originally, these cell culture tests were done in 96 well plates, but increasing use is now being made of 384 and 1536 well plates.



What is Cell and Tissue Culture?

Tissue Culture is the general term for the removal of cells, tissues, or organs from an animal or plant and their subsequent placement into an artificial environment conducive to growth. This environment usually consists of a suitable glass or plastic culture vessel containing a liquid or semisolid medium that supplies the nutrients essential for survival and growth.

Organ Culture: The culture of whole organs or intact organ fragments with the intent of studying their continued function or development is called Organ Culture.

Cell Culture.: When the cells are removed from the organ fragments prior to, or during cultivation, thus disrupting their normal relationships with neighboring cells, it is called Cell Culture.


How Are Cell Cultures Obtained?

• Primary Culture: When cells are surgically removed from an organism and placed into a suitable culture environment, they will attach, divide and grow. This is called a Primary Culture.

• There are two basic methods for doing this. First, for Explant Cultures, small pieces of tissue are attached to a glass or treated plastic culture vessel and bathed in culture medium. After a few days, individual cells will move from the tissue explant out onto the culture vessel surface or substrate where they will begin to divide and grow. The second, more widely used method, speeds up this process by adding digesting (proteolytic) enzymes, such as trypsin or collagenase, to the tissue fragments to dissolve the cement holding the cells together. This creates a suspension of single cells that are then placed into culture vessels containing culture medium and allowed to grow and divide. This method is called Enzymatic Dissociation.


Subculturing

• When the cells in the primary culture vessel have grown and filled up all of the available culture substrate, they must be Subcultured to give them room for continued growth. This is usually done by removing them as gently as possible from the substrate with enzymes. These are similar to the enzymes used in obtaining the primary culture and are used to break the protein bonds attaching the cells to the substrate.

• Some cell lines can be harvested by gently scraping the cells off the bottom of the culture vessel. Once released, the cell suspension can then be subdivided and placed into new culture vessels. Once a surplus of cells is available, they can be treated with suitable cryoprotective agents, such as dimethylsulfoxide (DMSO) or glycerol, carefully frozen and then stored at cryogenic temperatures (below -130°C) until they are needed.

• Buying And Borrowing; An alternative to establishing cultures by primary culture is to buy established cell cultures from organizations such as the American Type Culture Collection (ATCC; www.atcc.org)

• More frequently, researchers will obtain (borrow) cell lines from other laboratories. While this practice is widespread, it has one major drawback. There is a high probability that the cells obtained in this manner will not be healthy, useful cultures. This is usually due to previous mix-ups or contamination with other cell lines, or the result of contamination with microorganisms such as mycoplasmas, bacteria, fungi or yeast.

•


What Are Cultured Cells Like?

• Once in culture, cells exhibit a wide range of behaviors, characteristics and shapes.

• Cell Culture Systems: Two basic culture systems are used for growing cells. These are based primarily upon the ability of the cells to either grow attached to a glass or treated plastic substrate (Monolayer Culture Sytems) or floating free in the culture medium (Suspension Culture Systems). Monolayer cultures are usually grown in tissue culture treated dishes, T-flasks, roller bottles, or multiple well plates, the choice being based on the number of cells needed, the nature of the culture environment, cost and personal preference. Suspension cultures are usually grown either:

• 1. In magnetically rotated spinner flasks or shaken Erlenmeyer flasks where the cells are kept actively suspended in the medium; 2. In stationary culture vessels such as T-flasks and bottles where, although the cells are not kept agitated, they are unable to attach firmly to the substrate. Many cell lines, especially those derived from normal tissues, are considered to be Anchorage-Dependent, that is, they can only grow when attached to a suitable substrate. Some cell lines that are no longer considered normal (frequently designated as

• Transformed Cells) are frequently able to grow either attached to a substrate or floating free in suspension; they are Anchorage-Independent. In addition, some normal cells, such as those found in the blood, do not normally attach to substrates and always grow in suspension.


Types of Cells

• Cultured cells are usually described based on their morphology (shape and appearance) or their functional characteristics. There are three basic morphologies:

1. Epithelial-like: cells that are attached to a substrate and appear flattened and polygonal in shape.2. Lymphoblast-like: cells that do not attach normally to a substrate but remain in suspension with a

spherical shape.3. Fibroblast-like: cells that are attached to a substrate and appear elongated and bipolar, frequently

forming swirls in heavy cultures. It is important to remember that the culture conditions play an important role in determining shape and that many cell cultures are capable of exhibiting multiple morphologies.

Functional CharacteristicsThe characteristics of cultured cells result from both their origin (liver, heart, etc.) and how well they

adapt to the culture conditions. Biochemical markers can be used to determine if cells are still carrying on specialized functions that they performed in vivo (e.g., liver cells secreting albumin). Morphological or ultrastructural markers can also be examined (e.g., beating heart cells). Frequently, these characteristics are either lost or changed as a result of being placed in an artificial environment. Some cell lines will eventually stop dividing and show signs of aging. These lines are called Finite. Other lines are, or become immortal; these can continue to divide indefinitely and are called Continuous cell lines. When a “normal” finite cell line becomes immortal, it has undergone a fundamental irreversible change or “transformation”. This can occur spontaneously or be brought about intentionally using drugs, radiation or viruses. Transformed Cells are usually easier and faster growing, may often have extra or abnormal chromosomes and frequently can be grown in suspension. Cells that have the normal number of chromosomes are called Diploid cells; those that have other than the normal number are Aneuploid. If the cells form tumors when they are injected into animals, they are considered to be Neoplastically Transformed.


Finding A “Happy” Environment

• it means an environment that, at the very least, allows cells to increase in number by undergoing cell division (mitosis). Even better, when conditions are just right, some cultured cells will express their “happiness” with their environment by carrying out important in vivo physiological or biochemical functions, such as muscle contraction or the secretion of hormones and enzymes.

• To provide this environment, it is important to provide the cells with the appropriate temperature, a good substrate for attachment, and the proper culture medium and incubator that maintains the correct pH and osmolality

• Temperature is usually set at the same point as the body temperature of the host from which the cells were obtained. With cold-blooded vertebrates, a temperature range of 18° to 25°C is suitable; most mammalian cells require 36° to 37°C. This temperature range is usually maintained by use of carefully calibrated, and frequently checked, incubators.

• Anchorage-dependent cells also require a good substrate for attachment and growth. Glass and specially treated plastics (to make the normally hydrophobic plastic surface hydrophilic or wettable) are the most commonly used substrates. However, Attachment Factors, such as collagen, gelatin, fibronectin and laminin, can be used as substrate coatings to improve growth and function of normal cells derived from brain, blood vessels, kidney, liver, skin, etc. Often normal anchoragedependent cells will also function better if they are grown on a permeable or porous surface. This allows them to polarize (have a top and bottom through which things can enter and leave the cell) as they do in the body.

• The culture medium is the most important and complex factor to control in making cells “happy”. Besides meeting the basic nutritional requirement of the cells, the culture medium should also have any necessary growth factors, regulate the pH and osmolality, and provide essential gases (O2 and CO2). The ‘food’ portion of the culture medium consists of amino acids, vitamins, minerals, and carbohydrates. These allow the cells to build new proteins and other components essential for growth and function as well as providing the energy necessary for metabolism.


• The growth factors and hormones help regulate and control the cells’ growth rate and functional characteristics. Instead of being added directly to the medium, they are often added in an undefined manner by adding 5 to 20% of various animal sera to the medium. Unfortunately, the types and concentration of these factors in serum vary considerably from batch to batch. This often results in problems controlling growth and function. When growing normal functional cells, sera are often replaced by specific growth factors.

• The medium also controls the pH range of the culture and buffers the cells from abrupt changes in pH. Usually a CO2- bicarbonate based buffer or an organic buffer, such as HEPES, is used to help keep the medium pH in a range from 7.0 to 7.4 depending on the type of cell being cultured. When using a CO2-bicarbonate buffer, it is necessary to regulate the amount of CO2 dissolved in the medium. This is usually done using an incubator with CO2 controls set to provide an atmosphere with between 2% and 10% CO2 (for Earle’s salts-based buffers). However, some media use a CO2-bicarbonate buffer (for Hanks’ salts-based buffers) that requires no additional CO2, but it must be used in a sealed vessel (not dishes or plates).

• Finally, the osmolality (osmotic pressure) of the culture medium is important since it helps regulate the flow of substances in and out of the cell. It is controlled by the addition or subtraction of salt in the culture medium. Evaporation of culture media from open culture vessels (dishes, etc.) will rapidly increase the osmolality resulting in stressed, damaged or dead cells. For open (not sealed) culture systems, incubators with high humidity levels to reduce evaporation are essential.

lecture4- monolayer cell cultures (1)

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