lecture6-maintenance and charecterization of cells

8/14/2019 Lecture6-Maintenance and Charecterization of Cells

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© 2009, Maulik Suthar

Maintenance and

Characterization of cells

Maulik P. Suthar




MEASURING PARAMETERS OF

GROWTH

(i) increasing the number of cells;

(ii) increasing the size of the cells; or

(iii) increasing the amount of intercellular substance.

Since the intercellular substance of a tissue is usually a secretedproduct of the cell, for example collagen, it can be considered, so tospeak, as an extra-cellular extension of cytoplasm




Cell counting

(i) Prepare the dishes in which the cells have been grown (this example is for 100-mmdishes),

(ii) Prepare a trypsin solution, either 0.25 or 0.1 % (see note I below) in Hanks’ balancedsalt solution (containing no Ca2+ or Mg2+).

(iii) Pour Hanks’ solution (no Ca2+ or Mg2+) into 50-ml tubes,

(iv) Remove the medium from the dishes and set aside.

(v) Wash with 10 ml of Hanks’; remove.(vi) Add 10ml of trypsin solution and leave for30sec-1.5 min (note i).

(vii) Remove the trypsin solution and let stand at room temperature for 2-3 min (note i).

(viii)Add the growth medium (which includes 5% calf serum), 10 ml. At this point the cellswill detach from the surface. In these days of very expensive serum we use theconditioned medium obtained from step

(iv) to inhibit trypsin instead of fresh growth medium,

(ix) Mix well using a sterile pipette, drawing the cell suspension up and down thepipette 5-10 times,

(x) Count in a haemocytometer (Figure 1), by depositing a few drops of cell suspensionunder the coverslip. Use the four corners to count cells, divide by 4 and multiply by1()4 to obtain cells/ml.

For instance, if you count 140 cells in the four corners:• 140/4 = 35; 35 x 104 = 3.5 x 105 cells/ml




Cell counting




Population Doublings

• The number of population doublings occurring in a culture in which

the cells are dividing mitotically can be determined by a geometric

progression expressed as y = x2n,

where

x = number of cells at the beginning of the growth cycle,y = number of cells at the end of the growth cycle,

n = number of population doublings.




Cell Viability

i. Dye exclusion• The dye exclusion test is based on the concept that viable cells do

not take up certain dyes, whereas dead cells are permeable tothese dyes. Trypan blue (0.4%) is the most commonly used dye, buthas the disadvantage of staining soluble protein.

• In the presence of serum, therefore, erythrosine B (0.4%) is oftenpreferred . Cells are enumerated in the standard manner using ahaemocytometer.

• Some caution should be used when interpreting results as theuptake of the dye is pH- and concentration-dependent, and thereare situations in which misleading results can be obtained.

•Two relevant examples are membrane leakiness caused by recenttrypsinization and freezing and thawing in the presence of dimethylsulphoxide.

• A colorimetric assay using the MTT assay is being increasingly usedboth to measure viability after release of cytoplasmic contents intothe medium from artificially lysed cells, and for microscopicvisualization withinthe attached cell




DNA Amount

• The amount of DNA per dish can be determined, for instance, at 24-hintervals after plating. It is also a very simple procedure; indeed, for someone t/ained in biochemistry, it is easier than counting the number of cells. Since the amount of DNA per cell is usually constant (in mammaliancells, 6 x 10~12 g/ dipSoid cell in GI), the amount of DNA per dish is anindirect measure of the number of cells.

• The G] amount of DNA in somatic cells is generally referred to as the InamounT Cells in S-phase or in G2 have increased amounts of DNA with

respect to G, cells, but, if a population is truly growing, the increase in totalamount of DNA per dish will go way beyond the error caused by theindividual variations due to the distribution of cells throughout the cell cycle.

• DNA amount is probably the method of choice for sol id tissues. Using theamount of DNA per cell given above, one can calculate (from the amount of DNA per//g) the number of cells per ^g of tissue. A rough

• (very rough) estimate is that 1 /ug of tissue contains 5 x 1()8 cells, but thisestimate will vary greatly with the type of tissue.

• A possible source of error is polyploidy, that is an increase in the amount of DNA per cell from 2n to 4n or even 8n, in which case one could have anincrease in the amount of DNA per dish without a concomitant increase inthe number of cells.

• This is not a frequent occurrence, but it can happen, for instance, in certain

pathological conditions, in response to certain drugs or when a largeproportion of cells is blocked in G2. There are several methods for determinin the amount of DNA in a culture dish or in a tissue.




DNA Amount

http://www.phnxflow.com/Introduction%20to%20Cell%20Cycle%20Analysis.pdf




DNA by the Diphenylamine Reaction

• The quantitative determination of DNA is made by analysis of thedeoxyribose content of the hot TCA extract in a colorimetric reactionwith diphenylamine. Since a cell contains about 5 pg of DNA, andbecause microgram amounts are required to develop a detectablecolor, 1 X 106 cells is the very minimum numberthat can beanalyzed.

• In the hot TCA extraction step, the DNA structure collapses;deoxyribosides are released as both 5'- and 3'-phosphate bonds arehydrolytically cleaved. Deoxy-ribose is jthen freed as glycosidiclinkages with purines are broken. Under theseconditions, the bondbetween pyrimidines and deoxyribose is not cleaved, so only onehalf the deoxyribose residues in DNA are available to react with the

reagent. The blue color develops in the reaction




DNA by the Diphenylamine Reaction

• Deoxyribose is hydrolyzed to levulinic acid, which forms a complex withdiphenylamine

• Diphenylamine purification. Diphenylamine can be recrystallized bydissolving the reagent-grade compound in boiling hexane or in 70 percentethanol. Upon cooling, a white crystalline product is obtained

• Diphenylamine reagent. One gram of purified diphenylamine is dissolved

in 100 ml of reagent-grade glacial acetic acid and 2.75 nil of reagent-gradeconcen-trated sulfuric acid. The reagent is prepared immediately beforeuse.’standard curve purified DNA between 10 and 200 fig is reacted with the^(icnylamine reagent to obtain a standard curve. The intensity of the bluecolorI’-developed is directly related to concentration.

• A straight line is obtained when the concentration of DNA is plotted againstthe absorbance.

• Deoxyribose in the range of 5 to SO ng can also be used to obtain astandard curve. Since only deoxyribose linked to the purine reacts, aconversion factor can be employed by considering that one molecule of 2-deoxyribose is equivalent to a purine nucleotide plus apyrimidine nucleotide.

• Thus, the DNA content would be 4.87 times the measureddeoxyribose.




RNA Amount

• For a given cell type, the amount of RNA ought to be constant. As withDNA, G2 cells will have roughly twice the amount of RNA as G, cells. As ameasure of cell number, however, RNA amount is less accurate than DNAamount because, in several conditions, cells can grow in size and increasetheir RNA amount without cell division

• Indeed, RNA amount is a good indicator of cell size, rather than cell

number.• Most of the cellular RNA that is measured by bulk chemical methods or individual cell histochemical methods is ribosomal RNA (rRNA ~85% of totalcellular RNA).

• Since rRNA forms a part of the ribosome, on which protein synthesis iscarried out, it seems logical that RNA amounts ought to be a reasonableindicator of cell size, a hypothesis that has been empirically confirmed.

• Classic methods for the determination of RNA amounts can be foundelsewhere. If one wishes to determine the amount of RNA in individual cells,one needs expensive equipment that, in addition, require technical expertiseto operate.

• I prefer computerized microspectrophotometry to flow cytofluorimetry, butboth are complex and unless one is a devoted cell biologist who loves tolook at single cells, my advice is that when these instruments are needed

one should seek collaboration. Flow cytometry has already been discussedin a previous book from this series .




RNA by the orcinol reaction

• The quantitative determination of RNA is made by reacting the ribose in themacromolecule with orcinol. Since a single cell contains between 10 and 20pg of RN A, 5 X 105 cells is the minimal number that can be used for theassay.

• In the acid-extraction procedure, only ribose linked in a glycoside bond withpurine is freed and available to react with orcinol. A green-colored complexformsbetween ribose and orcinol.

• Orcinol purification. Orcinol is purified by dissolving it in boiling benzene,decolorizing with charcoal, and crystallizing after adding hexane.• A white crystal-line product can be obtained in this manner.• Orcinol reagent. One gram of purified orcinol is dissolved and 0.86 g of

Fe(NH4)2SO4-6H2O in 25.0 ml of double-distilled water.• This is stored as a stock solution in the cold at 4°C. The working reagent is

prepared immediately before use by adding 5.0 ml of the orcinol solution to

85.0 ml of concentrated HC1; additional distilled water is added to bring thevolume to 100.0

• ml. Reaction.• A 1-ml sample is mixed with 3 ml of orcinol reagent and heated in a boiling-

water bath for 20 minutes. After cooling, the intensity of the green color matdevelops is read at 670 nm. Standard curve. Purified RNA at aconcentration ranging from 10 to 100 jug is measured at required nm




Protein Amount

• The same comments apply here as to RNA amount.

• The amount of cellular protein is a reasonable indicator of cell size,

but a poor indicator of cell number.




Protein by the Lowry Reaction

• The quantitative determination of protein is made by reacting theFolin-Ciocalteau reagent with the alkali-solubilized precipitate after the extraction of DNA and RNA. The blue color that develops is aresult of (1) biuret reaction of proteins with copper ion in alkali, and(3) reduction of tungstic reagent by the tyrosine and tryptophanpresent in the treated protein.

• Since a cell contains between 300 and 600 pg of protein, thereaction can be carried out using 0.5 to 1.0 X 10s cells.

Reagents• Reagent A: dissolve 0.5 g of CuS04 -5H2O and 1 g of sodium or

potassium tar-trate in 100.0 ml of water.• Reagent B: carbonate-copper solution. Mix 50.0 ml of 2 percent

Na2CO3 with 1.0 ml of reagent A. Discard after 1 day.• Reagent C: alkaline copper solution. Mix 50.0 ml of 2 percent

Na2C03 in 0.1 A’ NaOIl with 1.0 ml of reagent A. Discard after 1day.

• Reagent D: diluted Folin reagent. Dilute the Folin-Ciochalteaureagent to make it 1 N in acid.




Mitoses

•However, mitoses arefleeting; in most cells they last only 45 min and, unlessone looks at precisely the right moment, one may miss them. Furthermore,the duration of mitosis can increase in certain cells, especially intransformed cells.

• Everything else being equal, if the duration of mitosis in cell line A is twicethat of cell line B, the number of mitoses in A will also be twice that of B,although the two cell lines may grow at the same rates. In tissues, thenumber of mitoses per 1000 cells (the mitotic index) is a reasonablemeasure of the proliferating activity of a cell population, but not of its growth.

• For instance, in the crypts of the lining epithelium of the small intestine,there are many mitoses. Fortunately for us, the small intestine in the adultindividual does not grow, because, for

• every new cell produced in the crypts, one dies at the tips of the villi.• So, in any given cell population, one must distinguish between cell division

and increase in cell number. There are also technical problems. To beginwith, if we wish to determine the mitotic index of cells in culture a 22-mm2coverslip will have to be placed into the culture dish.

• Mitoses can then be counted directly on the coverslips after fixation andstaining .Staining and counting of mitoses directly on plastic surfaces is notadvisable. However, suppose that a wave of mitoses occurse 25-27 h after stimulation of a quiescent cell population with growth factors. One may miss

it, unless samples are taken practically every hour.




Mitoses

• More economic in terms of time and money is to add a drug that willarrrest cells in mitosis. One can then count the percentage of cellsthat accumulate in mitosis over a certain period of time.

• The three main drugs for this purpose are colcemid, colchicine andnocodazole. Colcemid and colchicine are very similar but the latter is more toxic.

• For mitotic arrest, the optimal concentration of colcemid is 0.16,wg/ml for human cells or 0.04-0.08 ug/ml for rodent cells.

• I like to leave the drug in for 4 h, then fix the coverslips. By dividingthe time period into 4-h blocks (for instance, 16-20 h; 20-24 h; 24-28h) after serum-stimulation, once should be able to get a pretty goodidea of the mitotic activity of a cell population.

• If cells are left in colcemid (and especially colchicine) for more than4 h, cell damage occurs with loss of mitotic figures. Nocodazoleoffers the advantage that it can be used for longer periods of time,16-24 h.’We use it at concentrations of 0.04-0.2 ,Mg/ml, andmitoses, clearly identifiable, continue to accumulate.

• Depending on the cell line, and up to 12-16 h, nocodazole arrest is

reversible (so is colcemid-arrest but only up to 4 h).




Cells Arrested in Mitosis by Nocadozole

(i) Coat the coverslips (four per 100-mm Petri dish) with poly-L-lysine(Sigma 3000 mol. wt) at 1 ing/ml dissolved in Hanks’ (calcium,magnesium freesolution). Leave for 24 h.

(ii) Remove the polylysine solution and allow the coverslips to dry.

(iii) Plate 5 x 10s cells per 100-mm dish in normal growth medium,each dish containing two polylysine-coated coverslips.

(iv) After 18 h remove the medium and add fresh growth medium plus0.1-0.2 ,wg/ml of nocodazole dissolved in dimethylsulphoxide(DMSO).

(v) Leave for the desired period of time and then fix using the methodgiven above and stain with Giemsa/Sorenson’s buffer.




The Phases of a Culture




The Growth Studies

• Populations of animal cells cultured in vitro increase in number asthe individual cell divides mitotically. Whether they are primary cells,cell strains, or establishedcell lines, multiplication

• begins only after a period of adjustment and stops when anumber isreached that is saturating for the system.

• If the logarithm of number of cells present at any time in the cultureis plotted as a function of time, a smooth growth curve of characteristic of culture system i sobained

• The growth curve for mammalian cells can be divided into threephases: the lag phase,the log phase or exponential phase, and theplateau phase.

• The lag phase usually varies from 24 to 48hours. In this phase, thecells do not divide but are in the process of adapting to the medium.When the cells begin to divide, the population enters thelogarithmics of the culture cycle in which the cell number isincreasing at a constant rate is period, which lasts 2 to 8 days, thepopulation at any time is composed will points in the division cycle.




The Growth Studies

• The population doubling time for cultured cells ranges from 12 to 48hours. Since cultures are usually started with 50,000 to 200,000cells, four or five population doublings occur during the culturecycle.

• A population of cells will stop dividing when an essential nutrient isdepleted or an inhibitory substance is produced. At this point theculture enters the plateau phase, which is characterized by the factthat a constant cell number prevails over a period of time.

• The constancy of cell number may result because division ceases inall cells or because some cells degenerate and die while otherscontinue to divide




Mathematical Growth Curve

• The growth curve for a population of animal cells Analysis of CellMultiplication in Populations cultured in vitro can be subjected to amathematical analysis. Three useful parameters can be calculatedthat define the cell population increases with time:

(1) the number of population doublings occurring throughout the growth

cycle,(2) the population doubling time when the cells are dividing in the

exponential phase, and

(3) the rate of growth in the exponential phase. The formulas andmethods for calculating these parameters are presented in the

following discussion.




The Lag Phase

• This is an early phase in which there is no apparent increase in cellconcentration. This phase is associated with the cellular synthesis of growth factors I which may be required to reach a criticalconcentration before growth takes place.

• The length of this phase is dependent upon the culture mediumformulation as well as the initial concentration and state of the cells.The lag I phase tends to be longer at low inoculation densities or if

the viability of the inoculated cells is low.• This may occur if subculture is delayed. If a high density of cells with

good viability is inoculated into the culture medium then the lagphase may be eliminated altogether. Transformed cells have a Ilower requirement for growth factors and often show no lag phaseeven I when inoculated at lower concentrations

• In some circumstances, there may be a requirement to inoculate ata low I cell density, for example during cell cloning.

• Cloning is the establishment of a culture from a single cell. Thisensures that all cells in the culture are identical, i.e. a homogeneouspopulation. In this situation cell growth can be enhanced by adding afeeder layer which consists of irradiated cells incapable of growth

but metabolically active and capable of releasing growth factors intothe medium.




Exponential Phase




The stationary Phase

• This phase occurs when there is no further increase in cellconcentration. During the stationary phase, death rate = growth rate.

• At this point cell growth is limited by one of a number of conditionsnutrients may have been depleted to a level that cannot supportfurther cell growth;

• the accumulation of metabolic by-products may have reached alevel which is inhibitory to cell growth; the cells may have formed acomplete cover over the growth surface.

• Growth may stop when a single monolayer of cells covers theavailable substratum (called ‘confluence’).

• This phenomenon is associated with cell-cell interaction .

• During the stationary phase the cells may be metabolically activeeven though growth is not occurring. For example, in some caseshigh productivity of secreted proteins may occur during this phase.




The Decline Phase

• This phase occurs as a result of cell death. Cell viability is lowestduring the decline phase of culture - shown by a large differencebetween total and viable cell counts

• The measured viable cell concentration decreases as the cells lyseand their intracellular metabolites are released into the growth

medium.• There are two possible mechanisms of cell death in culture -

apoptosis or necrosis.




In vitro

•In exponentially growing cell populations the growth fraction is close to100% and gives very little information. Thus, if we take almost any cellline that grows with a doubling time of 24 h or

• less during the exponential growth phase, and we label it with[3H]thymidine for 24 h, the number of labelled cells would be close to100%.

• The odd unlabelled cells can probably be considered as dying cells. Thefraction of labelled cells becomes important in experiments dealing withthe effect of growth factors on the state of quiescence of a cellpopulation.

• For instance, many cell lines stop proliferating when they reachconfluence. It is desirable to test the extent of quiescence in a cellpopulation in culture by exposing the cells to [3H]thymidine. The interval

usually taken, more for expediency than for any logical reason, is 24 h.• Populations of cells that can be made quiescent should, at this point

have very low labelling indexes, that is not more than 1% of the cellsought to be labelled by a 24-h exposure to [3H]thymidine .

• At this point if one wishes to determine the effect of growth factors onthe proliferation of this particular cell population, all one has to do is to

add the growth factors together with [3H]thymidine and let theexperiment run for 24 or 48 h.




Flow of Cells Through the Cell Cycle

• At times it is desirable to determine whether there is a block or adelay in a particular phase of the cell cycle. This can be caused by adrug, by treatment with inhibitory factors or, for instance, intemperature-sensitive mutants, by a defect in one of the geneproducts that are necessary for cell cycle progression.

(i) A block in S-phase, due to direct inhibition of DNA synthesis, can be

detected by exposing the cells at various times after the treatment,to a 30-min pulse of [3H]thymidine. An inhibition of DNA synthesiswill result in a quick decrease in the fraction of cells that can belabelled by the pulse exposure, usually within 30 min of treatment.

(ii) If, instead, the decrease in the fraction of cells that are labelled bypulse exposure to [3H]thymidine is delayed, then one shouldsuspect a block in Gj, or even later. For

• instance, suppose that a given drug acts at a point in Gt which isroughly located 4 h before the S-phase. If that drug is given and thecells are then pulsed with

• [:’H]thymidine at various intervals after treatment, what one willobserve is that the percentage of labelled cells will remain constant,as in controls, for 4 h.




MAINTAINANCE OF CELL LINES




Common features of continuous cell lines

1. Smaller, more rounded, less adherent cells with a higher nucleus/cytoplasm ratio.

2. Increased growth ratio; cell doubling time reduced to 12-36 hour from 36- 48 hours for finite cell lines.

3. Aneuploid (between In and 4n) chromosome number showingconsiderable variation among cells of a cell line.

4. Reduced serum dependence.

5. Increased cloning efficiency.

6. Reduced dependence on substrate adhesion (anchorage) andincreased tendency to grow in suspension.

7. Many of the cell lines become malignant, but some cell lines are

normal and nonmalignant. The changes in growth control are under distinct positive (oncogen) and negative (tumour suppressor genes)control genes.

8. Ability to grow up to higher cell densities, i.e., growth is lessdependent on cell density.




Cryopreservation

• Freeze preservation of animal cells is now routine in allcell line banks. Acryoprotective agent like DMSO or glycerol is generally added to minimize injury to cellsduring freezing and thawing.

• Frozen ampoules are generally stored in liquid nitrogenrefrigerators which are rather convenient and quite safe.

• Following appropriate protocols most animal cells can bestored for quite long periods.




“Transformed” (or continuous) cell lines

• Large-scale production of bio-pharmaceuticals normallyutilizes “transformed” (or continuous) cell lines. Thesegrow faster than ordinary cells and can be grown atgreater densities, so they give higher yields inbioreactors.

• They are also easier to maintain in simple culture media.Like cancer cells, “transformed” cell lines do not stopdividing to repair any damage, instead, they keepdividing as long as there are sufficient nutrients and thatother suitable chemical and physical conditions for

survival are maintained.

Applications of Flow Cytometry to Cell




Applications of Flow Cytometry to Cell

Culture Identification and Control

Flow Cytometry to Cell Culture Identification



Flow Cytometry to Cell Culture Identification

and Control

lecture6-maintenance and charecterization of cells

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