11. Lecture 6 Animal Cell Biotechnology Cell culture storage and monitoring M.Butler. 1996. Animal cell culture and technology. Oxford:IRL Press. p 35
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15. Biomass Monitor Radio-frequency impedance can be measured by the probe. This is a measure of electrical capacitance (pF/cm) and correlates with the viable cell concentration. On-line measurement
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21. Coupled enzymatic assay 1: Lactate Pyruvate NAD + NADH + H + Absorbance at = 340 nm LDH 2: INT (tetrazolium salt) Formazan (red) Absorbance at = 490 nm diaphorase Promega kit
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25. Lecture 6 Animal Cell Biotechnology Cell culture storage and monitoring M.Butler. 1996. Animal cell culture and technology. Oxford:IRL Press. p 39
26. Lecture 6 Animal Cell Biotechnology Cell culture storage and monitoring M.Butler. 1996. Animal cell culture and technology. Oxford:IRL Press. p 39
Cells can be stored for long periods of time at low temperatures (-196 ºC). This allows you to maintain cell stocks without having to resort to primary tissue cultures. By using a cell banking system, you ensure that you have cells with uniform characteristics and growth characteristics. A typical Master Cell Bank might consist of 20-50 ampoules or vials, or more depending on the length of your project. Typically the master cell bank is undergoes quality control analysis. The maintenance of a master cell bank prevents the loss of a cell line due to contamination or genetic change that eventually occurs after a number of subcultures. Working Cell Bank consists of 100-200 ampoules; the wcb also undergoes quality analysis. Future cell samples for inoculation of cultures are removed from the working cell bank, while the master cell bank is accessed only when necessary.
Here is a variation of a banking system. You still have the upper two tiers, the MCB and the WCB. One ampoule from the working cell bank establishes the manufacturers working cell bank (up to 300 ampoules), which are taken out to initiate each production run. When the MWCB is exhausted, a new MWCB can be established using another ampoule from the working cell bank. Some of the ampoules at the MCB and WCB are removed for quality control, tested for contaminants, cell identity, etc.
Cells should be frozen while in the late exponential phase, to make sure they are strong enough to survive the freezing/thawing process. By insuring that only viable cells are frozen, you shorten the recovery phase. Glycerol and DMSO are commonly used, DMSO is the better of the two as it can penetrate the cell membrane more readily. Once membrane is made permeable, water flows out of the cell, such that ice crystals will form in the surrounding medium, but not in the cell.
Plastic box used to store vials.
Boxes containing vials can be stored in racks, which are lowered into the liquid nitrogen storage containers. Must check to make sure levels of liquid nitrogen don’t drop, must refill. May be a dipstick, or some sort of electronic alarm that indicates low levels of liquid nitrogen.
Liquid nitrogen can seep into improperly sealed vials. When the temperature increases, the liquid nitrogen becomes gaseous, vial may explode. Use methylene blue, sealed vials are dipped in the dye. If dye penetrates, the vial is discarded.
0.1 mm 3 = 0.0001 cm 3 = 0.0001 mL = 10 -4 mL. Count the total number of cells in 5 squares, divide by 5 and multiply by 10 4
Cells lyse in dilute solution, nuclei are stained by crystal violet and then counted.
This is a Coulter Counter - electronic cell counter for rapid counting of cells in suspension. Cells enter a hole and pass through a set of electrodes, interrupting the current between the electrodes. This sends a signal to the counter that a cell has passed through.
Exclude particles that are smaller than a cell – dust or cell fragments
Online monitoring system
Cells usually produce 100-500 pg of protein/mL. By determining the amount of protein in your medium you can get an estimate the total cell biomass. Can’t get cell numbers, as the protein content of a cell can vary during cell culture, not necessarily uniform. For DNA composition, use colored or fluorescent dyes that bind DNA.
This is a colorimetric assay that measures the amount of glucose in the culture medium, particularly for those systems where the cells are immobilized within a reactor and aren’t freely available for analysis. You know how much glucose was in the medium initially, you can determine how much glucose has been metabolized in a given period of time.
Also known as the MTT assay . It’s a sensitive, quantitative and reliable colorimeteric assay that can be used to measure the viability of cells. - MTT contains tetrazolium when cleaved results to colored product. Mitochondrial enzymes cleave a yellow colored, water soluble tetrazolium compound into a dark blue insoluble product. Percentage viability – percentage of population of viable cells. Viable concentration
Analysis of the release of intracellular enzymes can be used to monitor changes in cell viability. When cells are injured the membrane becomes more permeable, allowing for contents to leak out into the medium. The higher the rate of enzyme activity, the greater the loss of culture viability . Fairly active enzyme compared to other glycolytic enzymes Measures loss of viability -
Done either way
Denature protein with strong acid (TCA – trichloricacetic acid), protein will precipitate
Most precise of all methods, but time consuming
A = control = 12 B = treatment plate = 6 C = treatment plate = 4 Ability of cells to form a colony on each plate
Less precise method, extrapolate from linear portion of growth curve to time 0, compare derived count to original count. Sublethal Determine proportion of cells that has been effected by extrapolating back to day 0
After first point : establishes the common chromosome complement of a species or cell line (overhead diagram 1) Detects changes in cell cultures and cross contamination between cell lines. Chromosome identification aided by “banding” – use of a dye that generates a reproducible pattern of stained bands on each chromosome (overhead diagram 2). The most common is G-banding (Giemsa), bands formed are characteristic for each chromosome pair.
Stain – Giemsa stain – stains G bands on a chromosome – particular pattern on chromosome for cell lines Contamination on HeLa cells Nelson – Rees Involved in cancer work - looked at cancer cells, people were looking for virus in cancers (back then) nelson rees – g banding technique – patents in cancer cells had a HeLa pattern Labs were contaminating their cell lines with HeLa cells – which are fast growing
Isoenzymes are structural enzyme variants which catalyze the same reaction. When loaded onto a polyacrylamide gel, the isoenzymes will have different electrophoretic mobilities (each protein will move a certain length, depending on the size of the protein). After first point, put on overhead diagram. Isozyme banding pattern characteristic for each species. G6P dehydrogenase A-type – found primarily in black people
Antibody labelling: fluorescent antibodies bind to specific membrane antigens, the cells can then be identified using a fluorescent microscope or by a fluorescence-activated cell sorter. 4. DNA finger printing: DNA is digested and the fragments are separated by electrophoresis on an agarose gel. Radioactive probes are then hybridized to the fragments, forming a radioactive bar code that is unique for each cell line.
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