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%):

→ glycerol → dimethyl sulfoxide (DMSO)• cryoprotectants increase permeability of cell membrane

→ 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

Lecture 7 Animal Cell BiotechnologyCell culture storage and monitoring - conclusion

Tests to identify a cell line:1. Karyotype

2. Isoenzyme patterns

3. Antibody labelling

4. DNA fingerprinting

Lecture 7 Animal Cell BiotechnologyCell 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

Fig. 5.7

G-bands of a human X chromosome

Fig. 5.8

Lecture 7 Animal Cell BiotechnologyCell 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

Lecture 7 Animal Cell BiotechnologyCell 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