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Secondary cell culture

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ANIMAL CELL CULTURE TECHNIQUE

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Secondary cell culture

  1. 1. Secondary cell cultures  When a primary culture is sub-cultured, it becomes secondary culture or cell line. Subculture (or passage) refers to the transfer of cells from one culture vessel to another culture vessel.  Subculturing- Subculturing or splitting cells is required to periodically provide fresh nutrients and growing space for continuously growing cell lines. The process involves removing the growth media, washing the plate, disassociating the adhered cells, usually enzymatically. Such cultures may be called secondary cultures.
  2. 2.  Cell Line  A Cell Line or Cell Strain may be finite or continuous depending upon whether it has limited culture life span or it is immortal in culture. On the basis of the life span of culture, the cell lines are categorized into two types:  a) Finite cell Lines - The cell lines which have a limited life span and go through a limited number of cell generations (usually 20-80 population doublings) are known as Finite cell lines. These cell lines exhibit the property of contact inhibition(monolayer-cell growing), density limitation and anchorage dependence. The growth rate is slow and doubling time is around 24-96 hours.
  3. 3.  b) Continuous Cell Lines - Cell lines transformed under laboratory conditions or in vitro culture conditions give rise to continuous cell lines. The cell lines show the property of ploidy (aneupliody(abnormal chr no) )absence of contact inhibition and anchorage dependence. They grow in monolayer or suspension form. The growth rate is rapid and doubling time is 12-24 hours.  c) Monolayer cultures - When the bottom of the culture vessel is covered with a continuous layer of cells, usually one cell in thickness, they are referred to as monolayer cultures.  d) Suspension cultures - Majority of continuous cell lines grow as monolayers. Some of the cells which are non-adhesive e.g. cells of leukemia or certain cells which can be mechanically kept in suspension, can be propagated in suspension.
  4. 4.  Normal or transformed: Transformed cell lines usually have an increased growth rate and higher plating efficiency, are continuous, and require less serum in media, but they have undergone a permanent change in their phenotype through a genetic transformation.
  5. 5.  There are certain advantages in propagation of cells by suspension culture method.  These advantages are: (a) The process of propagation is much faster., (b) The frequent replacement of the medium is not required., (c) Suspension cultures have a short lag period, (d) treatment with trypsin is not required, (e) a homogenous suspension of cells is obtained, (f) the maintenance of suspension cultures is easy and bulk production of the cells is easily achieved., (g) scale-up is also very convenient.
  6. 6.  The cell lines are known by: a) A code e.g. NHB for Normal Human Brain. b) A cell line number- This is applicable when several cell lines are derived from the same cell culture source e.g. NHB1, NHB2. c) Number of population doublings, the cell line has already undergone e.g. NHB2/2 means two doublings.
  7. 7. COMMANLY USED CELL LINES  Cell lines are an invaluable scientific tool. They allow us to dissect the internal workings of tissues in a controlled environment without the ethical implications of working with whole organisms.  Starting with the first successful immortal cell line HeLa, the number of available cell lines has since diversified into a plethora(large amt) of options. Just like model organisms, the cell lines we work with define our scientific tribes.
  8. 8. Number 1: HeLa  Unlike the other cell lines above, HeLa is named after an individual, an American women called Henrietta Lacks. Shortly after establishment of this cell line, HeLa cells were used to proliferate the famous polio vaccine, and they continue to be the most widely used cell line in research labs worldwide.  According to the British newspaper The Guardian, HeLa “has led to hundreds, if not thousands, of new pieces of knowledge, and helped to shape the way medicine moved in the second half of the 20th century and the first decade of this one”.
  9. 9. Number 2: HEK 293  HEK293, or human embryonic kidney-derived epithelial cells, are arguably one of the most widely used cell lines in cell biology research. HEK293 is a rapidly dividing, robust line cell with a good reputation for post-translational modification of its heterologously expressed proteins.  Therefore, it’s hardly surprising that it is often the cell line of choice in transient and stable transformation experiments, for protein expression and production, and even in electrophysiological experiments.
  10. 10. Number 3: Insert your favorite immortal human cell line here!  Some may consider it cheating to use a cell line collection instead of a single cell line. But depending on your favorite object of study, you may end up using a collection such as those from the NCI-60 NCI-60 Human Tumor Cell Lines Screen (5).  Alternatively, you might be interested in working with Jurkat or HL-60 (white blood cells), MCF-7 (breast cancer), Saos-2 cells (bone cancer), PC3 (prostate cancer) or many others.
  11. 11. Number 4: Chinese hamster ovary cells  Chinese hamster ovary cells (CHOs) are clearly ovary- derived cells, but this time, we are taking mammalian cells. Similarly to Sf9 cells, they can exist both as adherent or suspension cells in culture.  CHO cells are used in various applications such as recombinant protein production and studies of the epidermal growth factor receptor .
  12. 12. Number 5: Sf9 insect epithelial cells  Derived from the ovaries of the fall armyworm moth (Spodoptera frugiperda), these cells are probably related to all insect cell lines in labs worldwide . Sf9cells can be cultured as adherent or suspension cells. Most cell lines are adherent cells, which grow only on the surfaces of culture vessels.  This limits the amount of cells you can expect to obtain from each culture. Similarly to E.coli, suspension cells can grow in the entire volume of the medium, thus increasing the amount of cells that can be harvested from a vessel.  Furthermore, and because of the high volume: cell number ratio, suspension cultures allow a much more effective use of medium than adherent cultures. Sf9/baculovirus systems are typically preferred for large-scale protein production including industrial manufacture of mammalian proteins, including the vaccine for cervical cancer CERVARIX® .
  13. 13. KARYOTYPE o The study of chromosomes, their structure and their inheritance is known as Cytogenetics. o Each species has a characteristic number of chromosomes and this is known as karyotype.
  14. 14. What each of the human chromosomes look like
  15. 15. In other words…  Chromosomes are digitally arranged so that they are matched with their homologue or “partner” chromosome.  Homologue chromosomes are the same size, shape, and carry the same genes, and one is inherited from each parent.  They are numbered according to size.
  16. 16. Sex determination with karyotype  This karyotype has 23 exact pairs, which means the person is female.  Note that #23 chromosomes are both X.
  17. 17. Normal human male  Note that #23 chromosomes are X and Y.
  18. 18. Is this person female or male?
  19. 19. Trisomy 21  Abnormality shown in karyotype  Note that there are three copies of #21 chromosome.  This person has Down Syndrome.
  20. 20. Monosomy X  Abnormality shown in karyotype  Note this person only has 1 copy of the X chromosome.  This female has Turner’s syndrome.
  21. 21. XXY Male (Extra X)
  22. 22. How are DNA samples obtained for karyotypes?
  23. 23. Amniocentesis: obtaining amniotic fluid which has cells from the fetus
  24. 24. Chorionic villi sampling: removing cells from the chorion with fetal tissue
  25. 25. Characterization of a cell line  Characterization of a cell line is vital for determining its functionality and in proving its authenticity as pure cell line.  Special attention must be paid to the possibility that the cell line has become cross-contaminated with an existing continuous cell line or misidentified because of mislabeling or confusion in handling DNA profiling.
  26. 26.  The various important factors for cell line characterization are:  It leads to authentication or confirmation that the cell line is not cross-contaminated or misidentified  It is confirmation of the species of origin  It is used for correlation with the tissue of origin, which comprises the following characteristics:  Identification of the lineage to which the cell belongs  Position of the cells within that lineage (i.e., the stem, precursor, or differentiated status)  For determination whether the cell line is transformed or not:  Whether the cell line is finite or continuous?  Whether the cell line expresses properties associated with malignancy?  It indicates whether the cell line is prone to genetic instability and phenotypic variation  Identification of specific cell lines within a group from the same origin, selected cell strains, or hybrid cell lines, all of which require demonstration of features unique to that cell line or cell strain
  27. 27. Parameters of Characterization Biochemical  Enzymes: Three parameters are available in enzymatic characterization:  The constitutive level (in the absence of inducers or repressors)  The induced or adaptive level (the response to inducers and repressors)  Isoenzyme(aa seq diff) polymorphisms(any forms)  Intermediate filament proteins : These are among the most widely used lineage or tissue markers. Glial fibrillary acidic protein (GFAP) for astrocytes(star shape-Glial Cells- CNS) and desmin(muscle specific-protein) for muscle are the most specific, whereas cytokeratin(found in cytoskeleton of muscle) marks epithelial cells and mesothelium.
  28. 28. GENETIC  Unique Markers: Unique markers include specific chromosomal aberrations (e.g., deletions, translocations, polysomy), major histocompatibility (MHC) group antigens (e.g., HLA in humans), which are highly polymorphic, and DNA fingerprinting or STR DNA profiling.  Transformation: The transformation status forms a major element in cell line characterization.
  29. 29.  Chromosome Content: Chromosome content or karyotype is one of the most characteristic and best-defined criteria for identifying cell lines and relating them to the species and sex from which they were derived. Chromosome analysis can also distinguish between normal and transformed cells because the chromosome number is more stable in normal cells (except in mice, where the chromosome complement of normal cells can change quite rapidly after explantation into culture).  Chromosome Banding: This group of techniques was devised to enable individual chromosome pairs to be identified when there is little morphological difference between them. For Giemsa banding, the chromosomal proteins are partially digested by crude trypsin, producing a banded appearance on subsequent staining. Trypsinization is not required for quinacrine banding(visualizing condensed chr). The banding pattern is characteristic for each chromosome pair.
  30. 30.  Chromosome Analysis  The following are methods by which the chromosome complement may be analyzed:  Chromosome count : Count the chromosome number per spread for between 50 and 100 spreads. (The chromosomes need not be banded.)  Karyotype : Digitally photograph about 10 or 20 good spreads of banded chromosomes. Image analysis can be used to sort chromosome images automatically to generate karyotypes.
  31. 31.  Chromosome counting and karyotyping allow species identification of the cells and, when banding is used, distinguish individual cell line variations and marker chromosomes. However, karyotyping is time- consuming, and chromosome counting with a quick check on gross chromosome morphology may be sufficient to confirm or exclude a suspected cross- contamination.

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