Application1. The study of basic cell biology, cell cycle mechanisms, specialized cell function, cell–cell and cell–matrix interactions. | 2. Toxicity testing to study the effects of new drugs. | 3. Gene therapy for replacing nonfunctional genes with functional gene-carrying cells. | 4. The characterization of cancer cells, the role of various chemicals, viruses, and radiation in cancer cells. | 5. Production of vaccines, mABs, and pharmaceutical drugs. | 6. Production of viruses for use in vaccine production (e.g., chicken pox, polio, rabies, hepatitis B, and measles). |
Explant Culture Procedure1. Obtaining the Explant | -obtained surgically using sterile equipment from mammals, rodents or avian organs or tissues | | | -ex 1: a piece of gingival tissue following tooth extraction can be removed as an explant to establish human gingival fibroblasts | | | -ex 2: a piece of adipose tissue can be used to establish mesenchymal stem cells | 2. Cut and Clean the Explant | -place the explant in a petri dish containing around 1-2 mL of incomplete medium (medium without serum) | | | -using a sharp surgical blade, you can cut it (usually around 1×1 mm pieces) | | | -collect the pieces of explant using a sterile forceps and wash gently | | | -washing can be done by transferring pieces into a centrifuge tube containing around 0.5 mL of incomplete medium | | | -gently mix by pipetting the medium 4 to 5 times, and allow the pieces to settle down and remove the upper medium | | | -can be repeated 2 or 3 times | 3. Culturing the Explants | -obtained explants are aseptically placed on a coated surface and allowed to attach to the surface in the presence of a rich culture medium | | | - medium ex: basal minimal media, Dulbecco’s Modified Eagle Medium (DMEM) or Minimum Essential Medium Eagle (MEM) supplemented with 10-15% serum | | | -cultured in standard tissue culture conditions (pH 7.2-7.4, temperature 37°C, 5% CO2 and humidity) to allow for cell migration and proliferation | | | -change the media every 3 days without disturbing the explants | | | -depending upon the health and age of the tissue, cells emerge out of the explant within 15-30 days | | | -once outgrowth of cells starts from the explant, add 5 mL of medium to the flask in subsequent days | 4. Once outgrowth of cells starts from the explant, add 5 mL of medium to the flask in subsequent days | -after the explants are completely surrounded by the cells, you can trypsinise the cells and subculture. | | | -it is better to use a lower concentration of trypsin (e.g. <0.25% of trypsin for 5 min) | | | -choose an appropriate size of flask for seeding, depending on the total number of cells obtained |
Pros and Cons of Types of Tissue Culture
Primary Culture-cultures prepared from tissues taken directly from animals | 1. Organ Culture | (google)-organ culture is able to accurately model functions of an organ in various states and conditions by the use of the actual in vitro organ itself | | | -maintenance of a piece of tissue, a part of organ or a whole organ in vitro | 2. Primary Cell Culture | -when taken tissue is dissociated, mechanically or enzymatically, into single cells which could be plated on a coated surface | 3. Slice Tissue Culture | -referred to as explant or organotypic cultures | | | -small pieces of tissue of interest are simply allowed to attach to an appropriate substrate and are cultured in enriched media | 4. Re-aggregate Culture | -dissociated cells is kept in suspension rather than allowed to settle on and attach to solid substrate | | | -cells tend to re-aggregate into small balls | | | -allowed cells cells to develop in three dimensions | 5. Histotypic or histoculture | -culture of intact tissues |
(Google) - Histotypic culture is defined as three-dimensional culture of one cell type, while the term organotypic implies the interaction of two or more cell types from a complex tissue or organ. Types of Cells1. 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. |
Types of Cell Culture1. Primary Cell Culture | -Adherent Cell Culture | | | -Suspension Cell Culture | 2. Secondary Cell Culture | - | 3. Cell Line | -Finite Cell Line | | | -Continuous Cell Line |
1. Primary Cell Culture-maintenance of growth of cells in culture medium using suitable glass or plastic containers | -using the mechanical or enzymatic methods | -dissociated directly from the parental tissue (such as kidney, liver) | -they will attach, divide and grow | | | 2 types of primary cell culture depending upon the kind of cells in culture | a) Anchorage Dependent /Adherent cells | -require attachment for cell growth | | | -monolayer culture system | | | -usually derived from tissues of organs such as kidney where they are immobile and embedded in connective tissue | | | (google)-have to be detached from surface before being subcultured | | | (google)-growth limited to surface area | b) Suspension Culture/Anchorage Independent cells | -do not require attachment for cell growth/do not attach to the surface of the culture vessels | | | -all suspension cultures are derived from cells of the blood system because these cells are also suspended in plasma in vitro e.g. lymphocytes |
Pros and Cons of Primary Cell Culture
2. Secondary Cell Cultures-When a primary culture is sub-cultured, it becomes known as secondary culture or cell line. |
3. Cell Line-cell population derived from a primary culture at the first subculture | (google)-usually clonal, meaning that the entire population originated from a single common ancestor cell | -the term does not imply homogeneity or the degree to which a culture has been characterized | | | may be finite or continuous depending upon whether it has limited culture life span or it is immortal in culture | a) Finite Cell Lines | -cell lines which have a limited life span and go through a limited number of cell generations | | | -growth rate is slow and doubling time is around 24-96 hours | b) Continuous Cell Lines | -grow indefinitely | | | -cell lines transformed under laboratory conditions or in vitro culture conditions give rise to continuous cell lines | | | -growth rate is rapid and doubling time is 12-24 hours | c) Transformed Cell Line | -cell lines obtained from tumor cells | d) Clonal Cell Line | -cells could be cloned in continuous cell lines to obtain genetically homogenous population |
Pros and Cons of Finite Cell Lines
Pros and Cons of Continuous Cell Line
Difference of Normal and Transformed CellsNormal Cells | Transformed Cells | 1. Anchorage-dependent (except blood cells) | 1. Nonanchorage-dependent | 2. Density-dependent inhibition of proliferation | 2. No density-dependent inhibition of proliferation | 3. Mortal; Finite Cell Line | 3. Immortal; Continuous Cell Line | 4. Contact Inhibition; Monolayer Culture | 4. No Contact Inhibition; Multilayer Culture | 5. Dependent on external growth factor signals for proliferation | 5. May not need an external source of growth factors | 6. Greater retention of differentiated cellular function | 6. Typically loss of differentiated cellular function | | | -shorter population doubling time | | | -reduced substrate adhesion | | | -genetic instability (e.g. show heteroploidy and aneuploidy) |
Contact-Inhibition of Growth
Density-dependent Inhibition of Proliferation-reduction in proliferative activity that correlates with the attainment of confluency, that is，occupancy of all available attachment surface | -can occur before confluence is reached, and reflects diminished nutrient supply and the release of cell-derived factors (including waste products) into the medium | | | Saturation Density | -population density (cells/cm2) at the point when it reaches density-dependent inhibition of growth | | | -population density (cells/cm2) at the point when it reaches density-dependent inhibition of growth |
Cell Ageing in Culture-also known as In vitro cell senescence | -involve progressive alterations in a number of cell characteristics | | | Normal cell lines commonly have a finite lifespan, that is, they do not grow beyond a finite number of cell generations (population doublings). | -Eg, the lifespan of normal diploid fibroblasts is in the range of 50-70 population doubling. |
| | Transformed Cells-cancerous cells | -possess all six hallmarks of cancerous cells : | 1. Growth factor independency | 2. No response to growth inhibitors | 3. Evasion of apoptosis (Natural cell death) | 4. Can promote angiogenesis (the development of new blood vessels) | 5. Unlimited proliferation - rapid increase | 6. Invasive - tending to spread very quickly and undesirably or harmfully |
Immortalization-Cell lines that have unlimited lifespan arc termed immortal or, preferably, continuous | | | the term immortalized and transformed are not synonymous | Although infinite lifespan is generally considered to be a characteristic of transformed cells, not all continuous cell lines exhibit alterations in growth control attributed to cellular transformation. | | | Immortalized Cells | -not yet cancerous, but have sufficient mutations to be able to be passaged forever, unlike non-transformed, non-immortalized cells, which all have a finite passage number | -population of cells from a multicellular organism due to mutation, which can escape normal cellular senescence and keep undergoing division | -this kind of cells can grow in vitro for prolonged periods |
Cell Strain-describe a subcultured population selected on the basis of its expression of specific properties, functional characteristics, or markers |
Clonal Culture / Clonal Selection-clone | -establishment of a cultured cell population derived from a single cell |
Sub-culturing (or passage)-Transfer or transplant cells of an ongoing culture to a new culture vessel so as to propagate the cell population or set up replicate cultures for study. | -Subculturing or splitting cells is required to periodically provide fresh nutrients and growing space for continuously growing cell lines. | -Such cultures may be called secondary cultures (first subculture from primary culture) | | | Criteria for Subculturing | 1. Cell concentration: should not exceed 1 x 10^6 cells/mL for most suspension-growing cells | 2. pH: which is linked to cell concentration, and declines as the cell concentration rises | 3. Time since last subculture: should fit a regular schedule | 4. Cell production requirements: for experimental or production purposes |
Pros and Cons of Animal Cell CultureAdvantage | Disadvantage | 1. Controlled physiochemical environment (pH, temperature, osmotic pressure, O2, osmolarity etc.) | 1. Expertise is needed, so that behavior of cells in culture can be interpreted and regulated. | 2. Controlled and defined physiological conditions - nutrient concentration, cell to cell interactions, hormonal control. | 2. Need of expertise and technical skill for the development, and regular use of tissue culture. | 3. Homogeneity of cell types (achieved through serial passages)/ Homogenous genetic population | 3. Ten times more expensive for same quantity of animal tissue; therefore, reasons for its use should be compelling. | 4. Economical, since smaller quantities of reagents are needed than in vivo. | 4. Unstable aneuploid chromosome constitution. | 5. Legal, moral and ethical questions of animal experimentation are avoided. | 5. Cost factor is a major limitation. | 6. Cost effective screening assays | -Establishment of infrastructure, equipment and other facilities are expensive. | 7. Easy production of biopharmaceuticals | -It is estimated that the cost of production of cells is about 10 times higher than direct use of animal tissues. | 8. Available in adequate numbers to do chemical study | 6. Control of the environmental factors (pH, temperature, dissolved gases, disposal of biohazards) is not easy. | 9. Easy to add genes (transfection) or regulate protein levels (RNAi) | 7. The native in vivo cells exist in a three- dimensional geometry while in in vitro tissue culture, the propagation of cells occurs on a two dimensional substrate. | | | -Due to this, the cell to cell interactive characters are lost. | | | 8. The cell lines may represent one or two types of cells from the native tissue while others may go unrepresented. | | | 9. Tissue culture techniques are associated with the differentiation i.e. loss of the characters of the tissue cells from which they were originally isolated. | | | -This happens due to adaptation and selection processes while culturing. | | | 10. Continuous cell lines may result in genetic instability of the cells. | | | -This may ultimately lead to heterogeneity of cells. |
Growth Measuring Methods1. Direct Methods | 2. Cells | -Packed Cell Volume | | | -Cell count and viability | | | -Colony forming unit | | | -Optical density (OD) | 3. Tissues | -Fresh weight and dry weight | 4. Indirect Method | -Mostly used for large-scale cultures |
Growth Observing1. Increase in turbidity of cells | 2. Increase in size of tissues/ explants | -swelling | | | -curling | | | -proliferation | 3. Decrease in turbidity and size | -death | | | -apoptosis and necrosis | 4. Microscopic observation | -Stereoscope | | | -Inverted microscope | | | Necrosis is caused by factors external to the cell or tissue, such as infection. |
Characterization of Cell Linesa) growth rate | b) karyotyping (C11) |
Growth Curve-established taking into consideration the population doubling time, a lag time, and a saturation density of a particular cell line. | 1. Lag Phase | The time the cell population takes to recover from such sub culture, attach to the culture vessel and spread. | 2. Log Phase | In this phase the cell number begins to increase exponentially. | 3. Plateau Phase | During this phase, the growth rate slows or stops due to exhaustion of growth medium or confluency. |
Bacterial Growth Curve-Unicellular organisms divide by binary fission | -Each cell grows to full size, replicates its genetic material then divides into two identical daughter cells. | -By identical means, two cells divide into four, four into eight and so on, leading to an exponential increase in cell numbers: 1 → 2 → 4 → 8 →2^n | -If we were to plot the number of cells in a population against time, we would get an exponential curve | -Growth usually slows down due to: | a) supply of nutrients becoming exhausted | | | b) because metabolism leads to an accumulation of harmful waste substances | | | Lag Phase | -When an inoculum of bacteria is first introduced into some growth medium, it will probably require a period to adapt to its new surroundings | | | -When an inoculum of bacteria is first introduced into some growth medium, it will probably require a period to adapt to its new surroundings | | | -Eg, the carbon source in the medium is unfamiliar, the cells will need time to synthesise the necessary enzymes for its metabolism. | | | -Synthesize molecules needed for protein synthesis and enzymes required for cell division | | | -no net increase in bacterial numbers, however the cells are metabolically active. | Length of the lag phase depend on: | a) age and general health of the cells in the inoculum | | | b) conditions of bacteria before transfer into growth medium | | | c) content of the growth medium | Log (exponential) Phase | -When the bacteria have acclimatized to their new environment and synthesized the enzymes needed to utilize the available substrates, they are able to start regular division by binary fission. | | | -leads to the exponential increase in numbers | | | -under optimal conditions, the population of cells will double in a constant and predictable length of time, known as the generation (doubling) time. | | | -Cells are dividing at maximal rate | | | -Cells are most susceptible to the action of antibiotics and other deleterious agents | Stationary phase | -exponential phase is limited by environmental factors, and as the rate of growth slows down, the culture enters the next phase | | | -The levelling out of the growth curve does not mean that cell division has ceased completely, but rather that the increase due to newly formed cells is cancelled out by a similar number of cell deaths. | | | -Occurs when the number of viable cells stops increasing | | | -Due to nutrients being used up and/or toxic products accumulating from cell’s metabolism | | | -as the death rate increases, the overall numbers fall and we enter the final phase of growth. | Death (or decline) phase | -As cells die off and the culture is unable to replace them, the total population of viable cells falls. | | | -Exponential decrease in the number of viable cells |
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