Universiti Tunku Abdul Rahman Bachelor of Science (Hons) Biotechnology/ Biochemistry UDBB2224 & UDEE2234 Cell and Tissue

Culture Techniques Tutorial 1 July 2011 (Week 7) ANIMAL CELL CULTURE

1) Describe types of cell in culture. 1- Primary cell culture a culture of cells that have been obtained directly from their place or origin 2- Anchorage Dependent /Adherent cells- A cell that grows, survives, or maintains function only when attached to an inert surface, such as glass or plastic. Usually derived from tissues of organs such as kidney where they are immobile and embedded in connective tissue. 3- Suspension Culture/Anchorage Independent cells- A cell that has lost the need for anchorage dependence, which is essential for cell growth, division, and spreading. Usually derived from cells of the blood system because these cells are also suspended in plasma in vitro e.g. lymphocytes 2) Describe the phases and check points in the cell cycle and explain the regulation of the cycle. S DNA replication G1 - growth and metabolism G2 growth of structural elements Go resting Phase M Mitosis G1 checkpoint- determines if the cell will enter the following S phase. G2 checkpoint- determines if the cell will enter the M phase and requires the proper completion of DNA synthesis. Spindle assembly checkpoint- between metaphase and anaphase and requires the proper attachment of all the chromosomes to the spindles apparatus.

The regulation of the cycle is regulated by cyclins and CDC kinases. CDK is active upon the binding of cyclin. Cyclin expression rises and falls within one cell cycle and repeat again in next cell cycle. Once bond to the cyclin, the mitotic cyclin dependent kinase complex phosphorylates protein involved in the early stage of mitosis. Active mitotic cdk/cyclin stimulates the breakdown of the nuclear envelope, chromosome condensation and mitotic spindle formation. It also controls the spindle assembly checkpoint by activating the anaphase promoting complex. G1 cdk-cyclin complex controls the G1 checkpoint by phosphorylation of retinoblasma (Rb) protein. Phosphorylation of Rb by cyclin/cdk complex allows the Rb to detach from E2F and activated E2F. Activated E2F help the transcription of a number of gene products that are essential to trigger S phase. 3) Briefly discuss the working procedure in laminar flow. i) Swab down work surface and all other inside surfaces of laminar flow hood with 70 % alcohol ii) Bring media, etc from cold store or thawed from freezer, swab bottles with alcohol and place those that you will need first in the hood iii) Collect pipettes and place at the side of the work surface in an accessible position iv) Collect any other glassware, plastics, and instruments that you will need and place them close by. v) Slacken but do not remove caps of all the bottles about to be used vi) Remove the cap of your bottles into which you are about to pipette and the bottle that you wish to pipette from and place the caps open side uppermost on the work surface at the back of the hood and behind the bottle.

vii) Insert pipette in a bulb or pipette aid, pointing pipette away from you and holding it well above the graduations viii) Tilt the bottle towards the pipette so that your hand does not come over the open neck, withdraw the requisite amount of fluid and transfer it to the recipient flask. ix) Discard the pipette from the hood and place it into the pipette cylinder containing disinfectant x) Replace the caps on the bottles and flasks, tighten all caps once complete the operation and remove all solutions and materials no longer required from the work surface.

4) Discuss recommended procedures in monitoring a cell culture to avoid contamination. a) Examine the cells visually and with a microscope before each operation - A sudden change in pH ; pH yellow, pH purple - Cloudiness in the medium - Presence of particles that different from the cells o Bacteria shimmering granular/rods o Yeast - round/ovoid particles o Fungi thin filamentous mycelia b) Record the nature of contamination when one occurs c) When working with different cell lines in parallel, pay close attention to avoid crosscontamination

5) Serum is normally added to culture media to promote cell growth. List the disadvantages of serum in medium. 1) Inhibits growth of some cell types, e.g. epidermal keratinocytes 2) May contain some cytotoxic constituents. 3) Serum quality varies from batch to batch which requires costly and time-consuming tests for each batch 4) Some growth factors are inadequate for specific cell types. 5) When cell cultures are used for production of biochemicals, the serum interferes with downstream processing. 6) Supply of serum is always lower than its demand. 6) Define the terms below : a) Contact inhibition phenomenon which cell division is inhibited by cell-to-cell contact. When one cell contacts another cell as a result of increased cell density. It first ceases to move, then metabolizes more slowly and ceases to divide. b) Complete medium - a medium that has had all its constituents and supplements added and is sufficient for the use specified c) Passage number - number of times that the culture has been subcultured.

d) Cell lineage - progression of cells down a particular differentiation pathway toward a specific differentiated cell type 7) Outline the main requirements of cell line characterization. 1) Authentication 2) Confirmation of the species origin 3) Correlation with the tissue of origin 4) Determination of whether the cell line is transformed or not 5) Indication of whether the cell line is prone to genetic instability and phenotypic variation 6) Identification of specific cell lines within a group from the same origin, selected cell strains, or hybrid cell lines

8) Name two suitable cryoprotective agents and state the concentration (%) of cyroprotective agents used to preserve cells. - dimethyl sulfoxide (DMSO) and Glycerol - Concentration: 5% - 15%

9) Define primary culture and explain why crude trypsin is the most common enzyme used in tissue disaggregation. Primary culture - Stage of the culture after isolation of the cells but before the first subculture Crude trypsin - (i) it is tolerated by a variety of cells (ii) It is effective for many tissues and (iii) its residual activity is neutralized by serum of the medium or by a trypsin inhibitor ( e.g. soybean trypsin inhibitor) in case of a serum free medium 10) Name two cell counting methods used for quantitation. - Hemocytometer - Electronic counting - Stained Monolayer