Precision of reading CHO DG44 cells NS0 cells - Techno Plastic

Swiss Federal Institute of Technology Lausanne, Faculty of BasicSciences, Institute of Biological and Chemical Process Sciences

A Novel Disposable Microtube for Rapid Assessment of Biomass in Cell CulturesMartin Jordan

ConclusionCells are pelleted into the capillary bycentrifugation for 1 minute at 2500 g.

The range is 0,1 - 2 million cells per ml(for 1 ml sample).

Reading errors (height of cell pellet) can bereduced to a relative S.D. of 1%.

The overall accuracy competes with thebest currently available techniques.

Features of microtube• Compatibility with standard rotors

• Sample volume is 1 ml or less

• Capillary is calibrated in µl

• Sensitivity fits cell culture needs

What is PCV?Measuring PCV or packed cell volume isa rapid method to quantify cells in smallsamples of blood. Due to the high celldensity in blood a few µl of samplecentrifuged within a glass capillary aresufficient to measure PCV. The densityof cell cultures is usually lower and muchlarger sample volumes are needed (e.g.10 ml for existing glass PCV tubes). PCVof 1% = 10 ml of pelleted cells per liter ofmedium.

Questions and answersWhich parameters affect PCV? The main parameters are centrifugation speed (Fig. 1) and centrifugation time (Fig. 2). Among other importantparameters are temperature and osmolarity.Can PCV-microtubes be used to count cells? PCV is an absolute volumetric value. Values can be converted into cell density (cells/ml). The conversionfactor has to be determined for a given average cell diameter.What are the advantages of PCV? No dilutions, manipulations, or calibrations are necessary. PCV represents an absolute value. The method works for allcell lines. It is reproducible and rapid; just sampling and a 1 minute centrifugation are needed.How is PCV calculated? The volume of packed cells is divided by the sample volume (µl/µl) and expressed as %.Cand PCV be used for aggregated cells? Yes, aggregates of less than 500 µm diameter can enter the capillary and form a compact cell pellet.Represents precision of reading the precision of method? Rather than errors due to inprecise readings we faced sampling and pipetting errors and effectsof other factors influencing cell size.

5 µl

4 µl

3 µl

2 µl

1 µl

1,6 mio

1,2 mio1,47 mio cells

Red lines:calibration for CHO DG44

Fig. 1: CHO cells were centrifugated for 1 minute atdifferent speeds (Eppendorf 5417C centrifuge withA-8-11 rotor). The experiment was done twice (seriesA and B,) and each point was the average from 2tubes.

Effect of centrifugation speed Effect of centrifugation time

Fig. 2: CHO cells were centrifuged for differentperiods of time at 2 different speeds (n=4). Forsubsequent experiments centrifigation for 1minute at 5000 rpm (2380g) was used.

Precision of reading

CHO DG44 cells NS0 cells

Comparison with other methodsSerial dilutions of two different cell lines werecarefully prepared and analysed by 3 differentmethods. Manual counting using trypan blue,automated counting (Casy), and the centrifugationmethod (1 tube per dilution, neither the dilution nor thesample identification was known by the experimenter).PCV was calibrated against cell number obtained bymanual counting.

Fig. 3A: PCV tubes compared with two othercounting methods. Note: Casy values wereunderestimated due to the presence of smallaggregates.

PCV tubes with 500µl sample volume

Fig. 3B: PCV tubes compared with two othercounting methods. For the two cell lines, PCVhad the best correlation coefficient.

Note: all this work was done with a prototype of a new product that will be manufactured by TPP (Techno Plastic Product AG, Trasadingen,Switzerland). Prototypes missed the scale that will be on the final product.

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