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
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Lecture 6 Animal Cell BiotechnologyCell 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
Cartwright, T. 1994. Animal cells as bioreactors. Cambridge:Cambridge University Press. p133
Lecture 6 Animal Cell BiotechnologyCell culture storage and monitoring
Cryopreservation in liquid nitrogen• stored in 1-2 mL plastic vials or glass ampoules
→ 107 cells/mL, just prior to stationary phase
• suspended in growth medium or serum supplemented with a cryoprotectant (5-10%):
→ minimize ice crystal formation that can damage cell membranes and organelles
Lecture 6 Animal Cell BiotechnologyCell culture storage and monitoring
M.Butler. 1996. Animal cell culture and technology. Oxford:IRL Press. p 24
Lecture 6 Animal Cell BiotechnologyCell culture storage and monitoring
M.Butler. 1996. Animal cell culture and technology. Oxford:IRL Press. p 24
Lecture 6 Animal Cell BiotechnologyCell culture storage and monitoring
• want slow freezing and fast thawing to maintain viability of stored cells
• ampoules frozen at -700C overnight → initial freezing of ~10C/min → ampoules are then placed directly in liquid nitrogen,
stored at -1960C, 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 370C water bath
• may explode if improperly sealed and liquid nitrogen penetrates seal
Lecture 6 Animal Cell BiotechnologyCell 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 mm2 (area) x 0.1 mm deep = 0.1 mm3 (0.1 μL) volume)
• cells/mL = total count (in 5 squares) x 104/5
Cell counting methodsCell counting methods
Lecture 6 Animal Cell BiotechnologyLecture 6 Animal Cell BiotechnologyCell culture storage and monitoringCell culture storage and monitoring
M.Butler. 1996. Animal cell culture and technology. Oxford:IRL Press. p 33
Lecture 6 Animal Cell BiotechnologyCell 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
Coulter counterFig. 5.3
Lecture 6 Animal Cell BiotechnologyCell culture storage and monitoring
M.Butler. 1996. Animal cell culture and technology. Oxford:IRL Press. p 35
Lecture 6 Animal Cell BiotechnologyCell 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
Lecture 6 Animal Cell BiotechnologyCell 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
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
Lecture 6 Animal Cell BiotechnologyCell 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
Lecture 6 Animal Cell BiotechnologyCell 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
Lecture 6 Animal Cell BiotechnologyCell 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
Lecture 6 Animal Cell BiotechnologyCell 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
Coupled enzymatic assay1: Lactate Pyruvate
NAD+ NADH + H + Absorbance at = 340 nm
LDH
2: INT (tetrazolium salt)
Formazan(red)
Absorbance at = 490 nmdiaphorase
Promega kit
Lecture 6 Animal Cell BiotechnologyCell 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
ATPADPAMP
Lecture 6 Animal Cell BiotechnologyCell 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
• 3H-leucine or 35S-methionine for protein synthesis
• tritiated thymidine (3H-Thymidine) for nucleic acid sythesis
Lecture 6 Animal Cell BiotechnologyCell 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
Lecture 6 Animal Cell BiotechnologyCell culture storage and monitoring
M.Butler. 1996. Animal cell culture and technology. Oxford:IRL Press. p 39
Lecture 6 Animal Cell BiotechnologyCell culture storage and monitoring
M.Butler. 1996. Animal cell culture and technology. Oxford:IRL Press. p 39