Cells Culture Techniques Marta Czernik

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1 Cells Culture Techniques Marta Czernik

2 Why we need the cell/tissue culture Research To overcome problems in studying cellular behaviour such as: - confounding effects of the surrounding tissues - variations that might arise in animals under experimental stress Reduce animal use Commercial or large-scale production Production of cell material: vaccine, antibody, human growth hormone, insulin

3 Areas where cell culture technology is currently playing a major role Model systems for: Studying basic cell biology, interactions between disease causing agents and cells, effects of drugs on cells, process and triggering of aging & nutritional studies Toxicity testing: Study the effects of new drugs Cancer research: Study the function of various chemicals, virus & radiation to convert normal cultured cells to cancerous cells Virology: Cultivation of virus for vaccine production, also used to study there infectious cycle Genetic Engineering: Production of commercial proteins, large scale production of viruses for use in vaccine production e.g. polio, rabies, chicken pox, hepatitis B Gene therapy: Cells having a functional gene can be replaced to cells which are having non-functional gene Medicine: chromosomal analysis amniotic cells

4 Cell culture (in vitro assay) applications

5 History - In 1885, Wilhelm Roux maintained tissues from embryonic chicken in warm saline for days, establishing the principle of cell culture. - In1907, Ross Harrison first developed a frog tissue culture technique. He probably chose frog for two reasons being a cold-blooded animal, no incubation is required and tissue regeneration is fast in frog. - In the 1940 s and early 1950 s, cell culture techniques were advanced significantly, in part, to support research in virology, allowed preparation of purified viruses for mass production of vaccines. - In 1949, Alan Park developed a protocol of cryopreservation. - A famous cell line HeLa cells was derived from cervical cancer cells taken from Henrietta Lacks, who died in Interest in culturing human tissues started in 1950 s after it was demonstrated (by HeLa; Gey) that human tumor cells could give rise to continuous cell lines. - In 1955, Eagle formed the basis of a medium known as basal medium, Eagle or BME, which was replaced by Eagle s minimum essential medium (MEM).

6 Advantages of cell culture - Absolute control of physical environment (ph, temperature, osmotic pressure, CO2 and o2 tension) - Homogeneity of sample - Less compound needed than in animal models STOP

7 Limitation of cell culture - Hard to maintain - Only grow small amount of tissue at high cost - Dedifferentiation - Instability, aneuploidy

8 Initiation of Cell Culture Tissue dispersion Primary cell culture Subculture Stored Cell line Continuous cell line Stored

9 Type of cell culture Primary Culture A primary culture is that stage of the culture following isolation of the cells, but before the first subculture. They are usually more representative of the cell types in the tissue from which they were derived and in the expression of tissue-associated properties. From the outgrowth of migrating cells from an explant From tissue that is disaggregated mechanically or enzymatically Subculture allows the expansion of the culture (now known as a cell line) but may cause a loss of specialized cells and differentiated properties (de-differentiation). (+)Advantages: Usually retain many of the differentiated characteristics of the cell in vivo (represent the tissue of their origin with regard to their properties). (-) Disadvantages: Initially heterogeneous but later become dominated by fibroblasts. The preparation of primary cultures is labor intensive and time consuming. Can be maintained in vitro only for a limited period of time.

10 Type of cell culture Continuous Cultures Derived from subculture (or passage, or transfer) of primary culture Subculture (cell line) = the process of dispersion and re-culture the cells after they have increased to occupy all of the available substrate in the culture Usually comprised of a single cell type Can be serially propagated in culture for several passages There are two types of continuous cultures: 1) Cell lines 2) Continuous cell lines

11 1) Cell line Once a primary culture is subcultured (or passaged, or transferred), it becomes known as a cell line. This term implies the presence of several cell lineages of either similar or distinct phenotypes. Finite life, senesce after approximately thirty cycles of division Usually diploid and maintain some degree of differentiation. It is essential to establish a system of Master and Working banks in order to maintain such lines for long periods. To provide large amounts of consistent cells for prolonged used Finite cell lines vs. continuous cell lines Cells lines with limited culture lifespans are known as finite cell lines; cells grow through a limited number of cell generations. If a cell line transforms in vitro, it becomes a continuous cell line, which is capable of unlimited proliferation

12 2) Continuous Cell line Continuous cell lines Can be propagated indefinitely Generally have this ability because they have been transformed - tumor cells - viral oncogenes - chemical treatments. The disadvantage of having retained very little of the original in vivo characteristics Greater growth rate; higher cell densities Require lower concentrations of serum and are more easily maintained in simple media HOWEVER, the appearance of a continuous cell line is usually marked by an alteration in cytomophology (smaller cell size, less adherent, more rounded), an increase in heteroploidy (chromosomal variation among cells) and aneuploidy (divergence from the euploid donor karyotype) and a loss of tissue-specific markers.

13 Cell culture morphology Morphologically cell cultures take one of two forms: Growing in suspension (as single cells or small free-floating clumps) cell lines derived from blood (leukaemia, lymphoma) Growing as a monolayer that is attached to the tissue culture flask. cells derived from solid tissue (lungs, kidney), endothelial, epithelial, neuronal, fibroblasts

14 Adherent cells Adherent cells are anchorage-dependent and usually propagate as a monolayer attached to the substrate. The attachment is essential for proliferation Adherent cells stop proliferating once they become confluent (i.e., when they completely cover the surface of the substrate) a phenomena known as contact inhibition Some cells will die if they are left in the confluent state for too long.

15 FILM - FIBROBLASTS

16 Suspension cells Suspension cells can survive and proliferate without being attached to a surface anchorage independence Hematopoetic cells (derived from blood, spleen, or bone marrow) as well as some transformed cell lines and cells derived from malignant tumors can grow in suspension Easier to passage as no need to detach them As the suspension cells reach to confluency, aseptically remove 1/3 rd of medium and add fresh one

17 Type of the Cells Fibroblast-like Epithelial- like Lymphoblast - like Neuronal - like On the basis of morphology (shape & appearance) or on their functional characteristics. They are divided into four: Fibroblast like cells attached to an substrate appears elongated and bipolar Epithelial like attached to a substrate and appears flattened and polygonal in shape Lymphoblast like cells do not attach remain in suspension with a spherical shape Neuronal like cells attached to an substrate appears very elongated and fin.

18 Phases of cell growth Lag Phase at this stage the cells do not divide. During this period the cells adapt to the culture conditions and the length of this phase will depend upon the growth phase of the cell line at the time of subculture and also the seeding density. Logarithmic (Log) Growth Phase cells actively proliferate and an exponential increase in cell density arises. The cell population is considered to be the most viable at this phase, therefore it is recommended to assess cellular function at this stage. Each cell line will show different cell proliferation kinetics during the log phase and it is therefore the optimal phase for determining the population doubling time. Plateau (or Stationary) Phase cellular proliferation slows down due to the cell population becoming confluent. It is at this stage the number of cells in the active cell cycle drops to 0-10% and the cells are most susceptible to injury. Decline Phase cell death predominates in this phase and there is a reduction in the number of viable cells. Cell death is not due to the reduction in nutrient supplements but the natural path of the cellular cycle.

19 Factors affecting cell proliferation Promotion of cell proliferation - low cell density (leaves the cell with free edge) - signals from environment: Growth factors Inhibition of cell proliferation - density limitation: high cell density - contact inhibition: cell contact signals from environment: p53 gene product

20 Factors affecting cell adhesion Cell adhesion is important for cell proliferation and differentiation (signaling through cytoskeleton) Cell adhesion molecule: Cell-cell interaction: CAMs, cadherin Cell-matrix interaction: integrin, transmembrame proteoglycan Tight junctional Tight junctional complex in epithelial cells for cell-cell interaction Enzymatic disaggregation digests the adhesion molecule and extracellular matrix Most cells from solid tissues grow as adherent monolayer Matrix-coated surface promotes cell proliferation and differentiation

21 Factors affect cell culture success Appropriate cells Suitable environment Solid phase substrate or phase on which the cell grow eg. glass, plastic, collagen, agar Liquid phase physicochemical and physiological constitution of the medium Gaseous phase Temperature Aseptic environment

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