Lecture 6 cell culture monitoring
- 1. Lecture 6 Animal Cell Biotechnology Cell culture storage and monitoring Cell storage and maintenance - Two-tiered cell bank master cell bank – storage of cells at early passage and established soon after receiving the original cell -> accessed only when absolutely necessary working cell bank – store of cells by growth for several passages of one of the master bank samples
- 2. Cartwright, T. 1994. Animal cells as bioreactors. Cambridge:Cambridge University Press. p133
- 3. Lecture 6 Animal Cell Biotechnology Cell culture storage and monitoring Cryopreservation in liquid nitrogen stored in 1-2 mL plastic vials or glass ampoules -> 10 7 cells/mL, just prior to stationary phase suspended in growth medium or serum supplemented with a cryoprotectant (5-10%): -> glycerol -> dimethyl sulfoxide (DMSO) cryoprotectants increase permeability of cell membrane -> minimize ice crystal formation that can damage cell membranes and organelles
- 4. Lecture 6 Animal Cell Biotechnology Cell culture storage and monitoring M.Butler. 1996. Animal cell culture and technology. Oxford:IRL Press. p 24
- 5. Lecture 6 Animal Cell Biotechnology Cell culture storage and monitoring M.Butler. 1996. Animal cell culture and technology. Oxford:IRL Press. p 24
- 6. Lecture 6 Animal Cell Biotechnology Cell culture storage and monitoring want slow freezing and fast thawing to maintain viability of stored cells ampoules frozen at -70 0 C overnight -> initial freezing of ~1 0 C/min -> ampoules are then placed directly in liquid nitrogen, stored at -196 0 C , stable almost indefinitely (80-90% recovery) -> can have programmable coolers to control rate of cooling to thaw, ampoules are quickly transferred from liquid nitrogen storage to 37 0 C water bath may explode if improperly sealed and liquid nitrogen penetrates seal
- 7. Lecture 6 Animal Cell Biotechnology Cell culture storage and monitoring 1. Hemocytometer microscope slide with a grooved calibrated grid cell suspension applied between coverslip and grid 9 squares (each square = 1 mm 2 (area) x 0.1 mm deep = 0.1 mm 3 (0.1 μL) volume) cells/mL = total count (in 5 squares) x 10 4 /5 Cell counting methods
- 8. Lecture 6 Animal Cell Biotechnology Cell culture storage and monitoring M.Butler. 1996. Animal cell culture and technology. Oxford:IRL Press. p 33
- 9. Lecture 6 Animal Cell Biotechnology Cell culture storage and monitoring Count the nuclei crystal violet solution with citric acid -> cells lyse, nuclei stain purple caution – cells could be binucleated (growth stopped), nuclei concentration may be higher than cell concentration good for counting anchorage dependent cells simple and effective, but can be a laborious process
- 10. Coulter counter Fig. 5.3
- 11. Lecture 6 Animal Cell Biotechnology Cell culture storage and monitoring M.Butler. 1996. Animal cell culture and technology. Oxford:IRL Press. p 35
- 12. Lecture 6 Animal Cell Biotechnology Cell culture storage and monitoring 2. Coulter counter rapid and accurate counting of multiple samples of cells, less than 5% error predetermined volume (0.5 mL) of a cell suspension is forced through a small hole in a tube by suction cells or particles (cause a change in electrical resistance as they pass between two electrodes, one inside and one outside the glass tube a series of pulses is recorded as a signal
- 13. Lecture 6 Animal Cell Biotechnology Cell culture storage and monitoring can change the size threshold to exclude dust or cell fragments high concentration cell suspensions should be diluted to prevent two or more cells from passing through at the same time cell aggregates should not be present
- 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
- 17. Lecture 6 Animal Cell Biotechnology Cell culture storage and monitoring 1. Protein determination cell protein used as a measure of biomass (total cellular material) Lowry and Bradford methods most sensitive methods, colorimetric assays 2. DNA composition DNA content of diploid cells is usually constant commonly used for cells in a solid tissue Indirect methods of cell growth determination
- 18. Lecture 6 Animal Cell Biotechnology Cell culture storage and monitoring 3. Glucose determination cell growth monitored by changes in the concentration of key components of culture medium correlation between cell concentration and consumption of glucose can also follow lactic acid production or oxygen consumption
- 19. Lecture 6 Animal Cell Biotechnology Cell culture storage and monitoring 1. Tetrazolium assay colorimetric assay for viable cells measure of cellular oxidative metabolism tetrazolium is cleaved to a colored product (blue) by the activity of dehydrogenase enzymes, indicates high level of mitochondrial activity in cells color is proportional to the number of metabolically active cells variation in results and responses between cell lines convenient for the rapid assay of replicate cell cultures in multi-well plates Cell Viability Measurements
- 20. Lecture 6 Animal Cell Biotechnology Cell culture storage and monitoring 2. Lactate dehydrogenase determination loss of cell viability followed by an increase of extracellular enzyme activity in medium -> enzymes leak from damaged cell membrane lactate deyhydrogenase (LDH) is the most commonly measured enzyme
- 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
- 22. Lecture 6 Animal Cell Biotechnology Cell culture storage and monitoring 3. Adenylate energy charge interconversion of the three adenylate nucleotides in the cell: ** decrease in the value gives an early indication of loss of viability in a cell population ( < 0.7-0.9) measured by HPLC or luciferase-luciferin enzyme system
- 23. Lecture 6 Animal Cell Biotechnology Cell culture storage and monitoring 4. Rate of protein or nucleic acid synthesis incubation of intact cells in growth media with radioactively labeled amino acid or nucleotide 3 H-leucine or 35 S-methionine for protein synthesis tritiated thymidine ( 3 H-Thymidine) for nucleic acid sythesis
- 24. Lecture 6 Animal Cell Biotechnology Cell culture storage and monitoring 5. Colony-forming assay directly measure ability of cells to grow low concentration of cells allowed to attach and grow on the surface of a Petri dish each viable cell will divide and give rise to a colony or cluster of cells useful for cytotoxicity assays
- 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
- 27. Lecture 7 Animal Cell Biotechnology Cell culture storage and monitoring - conclusion Tests to identify a cell line: Karyotype Isoenzyme patterns Antibody labelling DNA fingerprinting
- 28. Lecture 7 Animal Cell Biotechnology Cell culture storage and monitoring - conclusion 1. Karyotype establishing the identification and distribution of chromosomes -> indicates species of origin of the cells -> also indicates whether cells are transformed (chromosome count may vary) or any chromosome damage has taken place
- 29. Fig. 5.7
- 30. G-bands of a human X chromosome Fig. 5.8
- 31. Lecture 7 Animal Cell Biotechnology Cell culture storage and monitoring - conclusion 2. Isoenzymes structurally different forms of the same enzyme -> catalyze same reaction, have different protein size, structure specific activity stains used to develop banding pattern of isozymes to form zymogram -> characteristic of a particular cell line
- 33. Lecture 7 Animal Cell Biotechnology Cell culture storage and monitoring - conclusion 3. Antibody labelling cell line identified using fluorescent-labeled antibodies specific for a membrane antigen -> identified using fluorescent microscope or by fluorescence-activated cell sorter 4. DNA fingerprinting DNA is digested with restriction endonucleases and separated by electrophoresis radioactive probes hybridize to specific restriction fragments, bands highlighted by autoradiography
Editor's Notes
- #2: 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.
- #3: 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.
- #4: 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.
- #5: Plastic box used to store vials.
- #6: 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.
- #7: 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.
- #8: 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
- #10: Cells lyse in dilute solution, nuclei are stained by crystal violet and then counted.
- #12: 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.
- #14: Exclude particles that are smaller than a cell – dust or cell fragments
- #17: Online monitoring system
- #18: 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.
- #19: 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.
- #20: 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
- #21: 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 -
- #22: Done either way
- #24: Denature protein with strong acid (TCA – trichloricacetic acid), protein will precipitate
- #25: Most precise of all methods, but time consuming
- #26: A = control = 12 B = treatment plate = 6 C = treatment plate = 4 Ability of cells to form a colony on each plate
- #27: 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
- #29: 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.
- #31: 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
- #32: 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
- #34: 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.