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5. Cell Culture

Cell Culture Protocol

Cell culture protocols are meant to ensure that culture procedures are carried out to the required standards. This is not only meant to prevent the contamination of the cells, but to also ensure that the researchers themselves are protected from any form of contamination.

Moreover, the nature of the work is expected to conform to the appropriate ethical guidelines. Therefore, before anything else, it is essential to ensure that the entire procedure conforms with both medical-ethical and animal- experiment guidelines. This is because going against such legislation and guidelines can result in heavy penalties and even shutting down of the laboratory.

Before any work starts, carry out the following procedure:

  • Ensure that the working are is sanitized (using 70 percent ethanol)
  • Always use a new pair of gloves. If a pair of gloves has to be used for another cell culture procedure, they should be sanitized using 70 percent ethanol and allowed to air dry.
  • Any equipment that had been taken out of the cabinet should also be sanitized to prevent any contamination
  • Such equipment as pipette, glass jars and plastics to be used for the procedure should be autoclaved

Although there are a wide range of culture media for cells, it is important to keep in mind that cell cultures, and particularly primary cell cultures are easily prone to contamination in addition to the risk of containing undetected viruses. For this reason, all material should be handled as potentially infectious  in order to avoid any infections.

In addition, for safety purposes, work on cell culture should be carried out in the appropriate laminar flow hood, where air is directed away from the researcher.

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5. Cell Culture

Counting Cells

Counting the number of cells in a suspension is a process that involves the use of a stain. For instance, when trypan blue is used, it penetrates the cell membrane of the dead cells, but not the living cells.

The cells are then gently expelled into a haemocytometer (contains the counting chamber) under the cover slip and observed under a microscope. Cells are then counted within a given number of squares for calculations.


Significance

This method is largely preferred due to the fact that it allows for cells to be suspended in a solution rather than being held in a solid media. Here, therefore, it becomes easier to manipulate the contents thereby preventing them from forming clusters. With cell suspensions, it’s also easier to observe single cells under the microscope.

In this case, it becomes possible to not only study the structure of the cells, but also get to observe how well they have differentiated; viewing dead and living cells under the microscope.

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5. Cell Culture

Cell Suspension

In culture methods, cell suspension refers to a type of culture where cells are suspended in a liquid medium.

To obtain single cells, a friable callus (small tissue that falls apart easily) is put in agitated liquid medium (agitation allows for gaseous exchange unlike solid medium), breaking it up. This allows for single cells to be released, which are then transferred to another fresh medium.

Cell suspension cultures have a big advantage over the stationary ones given that it allows for the cells to be uniformly bathed. Moreover, given that the medium tends to be agitated, it allows for aeration of the medium, providing gases to the cells. Given that the medium is a suspension, it also becomes easy to manipulate the contents of the culture. 

Like any other culture, suspension cell culture has to be under controlled conditions, proving the cells with an ideal environment to proliferate. Once they reach about 80 percent confluence, it is time to subculture in order to ensure continued proper growth.

* 80 percent confluence refers to the state where 80 percent of the culture surface is covered with the growing cells.

In some cases, the cells in suspension may adhere on to the plastic surface of the culture flask or even form clumps. In such cases, a pipette can be used to pick these cells and expel them on to the surface of the flask and therefore away from the plastic surface. This helps obtain single cells given that they are no adhere on to the plastic surface.

Red Blood Cell Suspension

Red Blood Cell Suspension

  • (left: without hemolysis) red blood cell suspension (0.5% sheep RBCs in saline), seems red and opaque.
  • (middle: without hemolysis) RBCs sedimented spontaneously for 60 min. Note that the supernatant is not colored.
  • (right: hemolysis) RBC suspension treated with the hemolysin of S. pyogenes at 37C for 30 min, become transparent by hemolysis.
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5. Cell Culture

Cell Culture Media

Blood agar plates are often used to diagnose infection. On the right is a positive Streptococcus culture; on the left is a positive Staphylococcus culture.

In cell culture techniques, cells (or tissues) are removed from a plant or an animal and introduced into a new, artificial environment that can support their proliferation (survival and growth).

Some of the requirements of such an environment for the proliferation of the cells include:

  • A substrate (source of nutrition)
  • Ideal temperature range (controlled)
  • Growth medium, and
  • Ideal pH among others

Here, we shall focus on the medium (cell culture media)

Although there are different types of culture media (for different types of cells) they are typically composed of:

  • Glucose
  • Amino acids
  • Vitamins
  • Inorganic salts
  • Attachment factors Etc.

There are two major types of culture media. These include:

Natural media – Natural culture media is composed of biological fluids that are naturally occurring. Although this type of media can be used for a range of cells, its biggest disadvantage is that it may lack the exact components required by given cells, which can greatly affect reproducibility.

Artificial media – Also referred to as synthetic media, artificial media refers to the type of media that is produced by adding such nutrients as vitamins, gases (oxygen and carbon dioxide) and protein among others. These organic and inorganic nutrients are added so as to meet the specific needs of given cells, and thus provide the ideal environment for their growth.

As such, they can be used for a number of purposes including:

  • Providing immediate survival of the cells
  • Allowing for prolonged survival of the cells
  • Allowing for indefinite growth of the cells
  • Providing for specialized functions

On the other hand, culture may be categorized as:

Selective media– This is a special type of media that only allows for certain cells to grow. For instance, blood agar (used to isolate Streptococcus & Moraxella species) can be turned in to a selective media by adding antibiotics.

Differential media– This type of media allows for different types of cells/microorganisms to grow depending on their metabolism.

As mentioned above, different types of synthetic media are prepared in a manner that will provide the ideal proliferation environment for given cells. For this reason, synthetic media can be divided in to four major categories.

These include:

Serum containing media – In these types of media, serum (fetal bovine serum) is used as a carrier for nutrients and growth factors among others that tend to be water insoluble.

Serum-free media – These types of media is typically produced for the purposes of supporting single cell type of culture. As such, it provides specified nutrients and other factors required by the cell type. In this media, serum is absent because it present some disadvantages and can result in misinterpretation of immunological results.

Chemically defined media – Like the name suggests, this type of media is composed of contamination- free pure organic and inorganic ingredients. Constituents of this type of media are typically produced through genetic engineering in bacteria/yeast.

Protein-free media – Protein- free media are typically lacking of any type of protein. It’s largely used to promote superior growth of the cells as well as protein expression in addition to facilitating for the purification of any expressed product.

Some of the major components of cell culture media include:

  • Nutrients – provided for by peptides and amino-acids, which are the building blocks of proteins
  • Carbohydrates for energy
  • Essential minerals  such as calcium, magnesium, phosphates and iron among others buffering agents such s acetates to stabilize the culture media
  • Vitamins
  • PH change indicators such as phenol red

Cell culture media are used for the proliferation of cells, which can then be identified and studied. As such, it can be used for various purposes including for education, diagnosis and treatment of a disease among others.

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5. Cell Culture

Primary Cell Culture

Cell culture is a process where cells (animal or plant cells) are removed from the organism and introduced in to an artificial environment with favorable conditions for growth. This allows for researchers to study and learn more about the cells.

There are three major types of cell culture, which include:

  • Primary cell culture
  • Secondary cell culture, and
  • Cell line

Here, we shall focus on primary cell culture.

There are two types of primary cells:

Adherent cells – Also referred to as anchorage dependent cells, these are the type of cells that require attachment for growth. Adherent cells are immobile, and obtained from such organs as kidney.

Suspension cells – These are the type of cells that do not require attachment in order to grow. They are therefore also referred to as anchorage independent cells, and include such cells as lymphocytes found in the blood system.

In primary cell culture, cells obtained from such parental tissues (living tissues) as the liver and kidney, are introduced into suitable media for growth. Once the cells have been obtained, they can either be cultured as explants culture, suspension or monolayer.

* In primary cell culture, the cells must have been obtained from the parental/living tissue. That is, they are not from another culture process.

Before the cells are cultured, they are first subjected to enzymatic treatment for dissociation. However, has to be for a minimal amount of time to avoid damaging or killing the cells. Once single cells are obtained, they are then appropriately cultured in media to allow them to grow (divide) are reach the desired numbers.

Initially, the culture tends to be heterogeneous in that it’s composed of different types of cells obtained from the tissue. Although this can be maintained through the in vitro process (in a culture in a suitable media) this would only be for a limited period of time.

Through the transformation process, the primary cells may be used for a long period of time, changing the culture over time. These cells are refers to as continuous cell lines.

However, primary cells are typically preferred over continuous cell lines because of the fact that they are more similar (physiologically) to in vivo cells (cells from the living tissue). In addition, continuous cell lines may undergo certain changes (phenotypic and genotypic changes) which would result in discrepancies during analysis. As such, they cannot be used to determine what is happening to the in vivo cells. It’s for this reason that primary cells are preferred.

Given that the primary cells significantly resemble the cells obtained from living tissue, they are important for research purposes in that they can be used to study their functions, metabolic regulations, cell physiology, development, defects and conditions affecting the tissue of interest.

Also, they are used for such purposes as vaccine production, genetic engineering drug screening as well as toxicity testing and prenatal diagnosis among others.

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5. Cell Culture

Significance of Cell Culture

Cell culture is an important technique in both cellular and molecular biology given that it provides the best platform for studying the normal physiology and biochemistry of cells. A cell is the basic structural, functional and biological unit of all living things.

In order to understand an organism or given tissues, it is important to understand how its cells work. Through cell culture, this becomes possible especially due to the fact the primary cells resemble the parental cells from the organism/tissue.

Whatever is learnt about the cells in vitro is representative of what is happening to the organism/tissue. This makes cell culture significantly important for vaccine development, screening (drugs etc) and diagnosis of given diseases/conditions.

Given that different types of cells require different environments for proliferation, there are different types of media used for culture such as serum-free media and serum containing media among others.

Once the right requirements have been provided, the cells will  increase in numbers and may form colonies, which can then be easily seen and identified. However, all this requires that the purpose of the procedure be understood.

Having a good understanding of what the procedure is meant to achieve, it becomes easier to prepare the culture with the right components. By understanding what the procedure is aimed for, the researcher will know whether to prepare a selective media (which allow for specific cells to grow) or differential media (allowing for different types of cells to grow).

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5. Cell Culture

Media supplements for cell culture

Media supplements help to optimize cell growth for specific applications depending on the chosen tissue or cell type. The advantages of using media supplements such as growth factors or cytokines are that they may improve cell viability and growth and keep cells healthier for longer. For example, fibroblast growth factor (FGF) plays important roles in diverse biological functions in vivo and in vitro and can maintain cell culture over the weekend as Proteintech thermostable FGF (HZ-1285) does not require media changes every day.

The stability of FGFbasic-TS and FGF basic (E. coli-derived) in xeno-free, chemically defined cell culture media at 37˚C. The protein concentration was determined by ELISA each day for 3 days. After one day of incubation at 37˚C, FGF basic was undetectable, while FGFbasic-TS was present at levels of 60%, 35%, and 20% of its starting concentration at days 1, 2, and 3.

Growth factors play an essential role to maintain the in vitro culture growth. Depending on the environment, certain cells can give rise to a variety of lineage-specific cell types. See below a summary of the key growth factors needed for normal cell growth, metabolism, cell development in culture and their differentiation process.

SupplementCat noInvolved in/used for
Fibroblast growth factor (FGF)HZ-1285Embryonic development, neuron differentiation, and the proliferation of cells of mesodermal origin and many cells of neuroectodermal, ectodermal, and endodermal origin.
Bone morphogenetic protein 2 (BMP-2) and bone morphogenetic protein 4 (BMP-4)HZ 1128HZ-1045Bone formation and regeneration.Uses as differentiationfactor of pluripotent stem cellsand promotes osteogenic differentiation of mesenchymal stem cells
Hepatocyte growth factor (HGF)HZ-1084Epithelial-mesenchymal transition.Acts as a mitogen for many cell types including hepatocytes
Granulocyte-macrophage colony-stimulating factor (GM-CSF)HZ-1002Promoting growth and differentiation of hematopoietic progenitor cells.Used to support the in vitrocolony formation of granulocyte-macrophage progenitors.
Activin AHZ-1138Regulating cell proliferation.Used to maintainstem cell pluripotency and self-renewal in vitro
Beta-nerve growth factor (beta NGF)HZ-1222Differentiation and survival of neurons
Stem cell factor (SCF)HZ-1024Promoting survival, expansion,and differentiation of hematopoietic stem cells.
Transforming growth factor beta (TGF-beta)HZ-1011HZ-1092HZ-1090Regulating cell growth, proliferation,and differentiation.Widely used, e.g.,in undifferentiated embryonic stem cellcultureand induced pluripotent stem cells
Vascular endothelial growth factor (VEGF121 and VEGV165)HZ-1204HZ-1038Angiogenesis and vasculogenesis.Used in endothelial cell culture.
Wnt3aHZ-1296Regulation of neuronal stem cell self-renewal.Used in expansion of neural stem cells
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5. Cell Culture

Cell culture environment

Cell culture is an amazing tool that allows for easy control and manipulation of all physiochemical and physiological cell factors, such as, temperature, osmotic pressure, pH, gas, hormones, and nutrients.

Cell culture environment.

MediapHTemperatureCO2
Contains nutrients, growth factors, and hormonesAverage pH for mammatian cells is pH 7.4Depends on body temperature of the hostControlled by media
Sera source of growth, tipids,hormones Mammalian cell lines 36-37°COrganic or C02 bicarbonate buffer systems are popular
  Insert cell lines 27-30°CCan impact pH
   4-10% C02 is most common
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5. Cell Culture

Aseptic techniques required while working with cell culture

To be successful in cell culture, it is essential to remain a contamination free environment (bacteria, fungi etc) Aspetic techniques ensure that no microorganisms enter the cell culture. Cell culture sterility is ensured by set of procedures.

Aseptic techniques required while working with cell culture.

HandlingReagents/MediaWorkplace
Slow/careful handling.Pre-sterilisation of all reagents/equipmentCell culture hood works properly
Sterilization of all items before starting.No contamination in reagents (expiration date, appearance normal).Frequent de-contamination (hood, fridge etc)
Sterile pipettes Work area: sterile and tidy
No touching of sterile items to non-sterilized surfaces 
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5. Cell Culture

Cell culture safety

Work in a cell culture laboratory is associated with different risk factors and hazards such as toxins or mutagenic reagents. The human/animal material may contain viruses and other dangerous biological agents. Therefore, while manipulating human/animal material it is important to follow general safety guidance for laboratory practices.

1. Wash hands when entering and before leaving the laboratory.

2. Wear safety clothes (gloves, closed shoes, lab coat).

3. No eating, drinking, smoking.

4. No or low aerosol creation.

5. Decontaminate all surfaces before and after the experiment.

6. Work in accordance with the facility guidelines.

7. Dispose of all waste in an appropriate way.

8. Restricted access to authorized personnel only.

9. Avoid using sharp objects.

10. Always clearly label all samples.

11. Report all incidents to the safety officer.