Appl Microbiol Biotechnol (1991) 36:208-210

Applied Microbiology Biotechnology © Springer-Verlag 1991

Short contribution A novel micromanipulation technique for measuring the bursting strength of single mammalian cells Z. Zhang 1, M. A. Ferenczi 2, A. C. Lush 3, and C. R. Thomas 1 i School of Chemical Engineering, The University of Birmingham, Edgbaston, Birmingham B15 2TT, UK 2 National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK z British Bio-technology Ltd., Watlington Road, Oxford, OX4 5LY, UK Received 16 May 1991/Accepted 11 July 1991

Summary. Information about the bursting strength o f animal cells is essential if the mechanisms of cell damage in bioreactors are to be understood, and if cell mechanical properties are ever to be related to cell structure and physiology. We have developed a novel cell compression technique that makes it possible to directly measure the bursting strength of single mammalian cells, and to infer information about cell mechanical properties.

modulus of the cell, but not the bursting strength. The main problem in measuring the bursting strength o f single mammaJian cells is the difficulty of measuring accurately the force required to cause a known large deformation o f a cell. A technique based on micromanipulation has therefore been developed to measure the bursting strength of single cells and, by modelling o f the cells (to be reported elsewhere), to derive other information such as the cell compressibility modulus and shear modulus.

Introduction

Materials and methods

Mammalian cells, such as hybridomas in suspension cultures, are generally considered susceptible to shear or other mechanical forces, owing to their lack o f a protective cell wall. Such forces occur during mixing and sparging o f bioreactors, especially if high cell densities and large scale are to be achieved, and the consequent potential for damage limits the design and operation o f cell culture fermentors. Furthermore, it is not possible to gain real insight into the mechanisms determining cell mechanical properties, and how the latter might be controlled, without the ability to make the appropriate measurements. Current indirect methods for studying the effects o f shear and other forces on mammalian cells in suspension culture (Stathopoulos and Hellums 1985; Smith et al. 1987; Petersen et al. 1988; Cherry and Kwon 1990) are unsatisfactory because the results of the experiments are difficult to interpret. A need therefore remains for a technique to directly measure cell mechanical properties (including bursting strength). Such a method could be the basis for systematic investigations on cultured animal cells (Bleim 1989). Cell aspiration techniques ( H o c h m u t h 1987; Sato et al. 1987) cause only small area changes to cell membranes, therefore giving the elastic compressibility modulus and shear

Experimental equipment. The basic principle of this technique is the capture and squeezing of a single cell between two parallel surfaces (Evans and Skalak 1980) using optical fibres with flat ends as probes. Figure 1 shows the squared ends of two such probes magnified 400 times. One probe was mounted on a threedimensional micromanipulator (Prior-Martock, Cambridge, UK). Another was connected to the output tube of a force transducer (Model 406A, Cambridge Technology, Watertown, MA, USA) by

Offprint requests to: C. R. Thomas

Fig. 1. Two optic fibres with flat ends, positioned for squeezing a cell. The flat ends were made by grinding optic fibres with diameters of about 50 ~tm. Using varnish, hundreds of such fibres were glued tightly into a hole in a stainless steel block with finished surfaces. The optic fibres were cut by a cleaving tool (Leetec, London, UK) to make preliminarily squared surfaces. The ends of the glued fibres were polished by lapping films in finishes of 0.3-3 lxm (Angula, Milton Keynes, UK). The varnish between the fibres and the block was then dissolved using toluene. Optic fibres with acceptably fiat surfaces were chosen for probes

209 low-melting-point paraffin. The force transducer was mounted on a second three-dimensional micromanipulator, a prototype of very fine motion of + 0.2 lxm accuracy (Prior-Martock). A heavy block of metal between the force transducer and the fine micromanipulator was used to damp down vibrations. Culture medium (40 Ixl) containing cells was put onto a small microscope slide by micropipette. The slide (16 mm long, 4 mm wide and 0.2 mm thick) was located on a copper stage held by a third micromanipulator. A 4-ram diameter hole in the centre of the copper stage allowed sample illumination from beneath. The temperature of the medium on the stage was kept at 37 +__0.5°C by controlling the surface temperature of the copper stage using resistance heating.

Cell line and culture media. Hybridoma TB/C3 cells from synchronous and continuous cultures were used in this study. For the synchronous culture, the basic medium consisted of RPMI 1640 (Sigma, Poole, UK) supplemented with 10% foetal calf serum. The culture was synchronized by the double thymidine block method (A1-Rubeai and Emery 1990). For the continuous culture, the medium consisted of RPMI 1640, supplemented with 5% newborn calf serum, meat peptone and Pluronic F-68. The cells were cultured in a 500-ml magnetically stirred flask, with a working capacity of 300 ml. The flow rate of air supplemented with 5% v/v CO2 was 100 ml min -1. Experimental procedures. The probes were aligned horizontally under a long working distance microscope (objective, x40; working distance, 6.8 mm) and were lowered into a drop of culture medium on a slide until they almost touched the top surface of the latter. A cell was chosen for testing, and the probes were moved until both sides of the cell were just in contact with the probe ends. A fine micromanipulator holding one probe was then moved a short distance, about 6 lxm, so that the cell was held tightly between the two surfaces (see Fig. 1). Before the cell was squeezed to bursting, the slide was lowered a few microns in order to ensure that the cell and probes did not touch the slide surface. The fine micromanipulator was then moved again so that the cell was squeezed within a pre-fixed period of time, usually 5.12 s. During squeezing of a cell, the force being imposed on it was measured by sampling the voltage signal from the force transducer, amplified tenfold, using a PC 30-D data acquisition board (Amplicon Liveline, Brighton, East Sussex, UK) fitted to a personal computer (Dell Computer Corporation, Austin, Tex., USA). When the two probes eventually touched, they were separated to return the imposed force to zero. From curves of force versus the distance between the two flat planes, which can be found from the time of motion, the bursting strength and the diameter of the cells were obtained, as discussed below.

Results and discussion When two flat surfaces hold a cell and gradually squeeze it, the cell deforms until it bursts. Figure 2 shows a typical experimental record o f force versus sampling time during such squeezing of a cell. The force measured rose f r o m A to B as the cell deformed. Point B corresponds to the cell bursting. The probes touched each other at point D, causing a considerable rise in the force exerted. U p to this point, the distance between the two parallel planes can be calculated from the sampling time, as the speed of the probes was preset (and previously calibrated). After the probes were separated, the force recovered to the base level, line FG. The bursting strength of a cell is calculated from the m a x i m u m change of force (the difference between B and line F G on the experimental curve). The cell diameter can be calculated f r o m the sampling time to point

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D, provided a correction for the initial holding squeeze (see experimental procedures) is applied. The speed o f the fine micromanipulator employed in this study was 3.94 Ixm s -1, which was slow enough for the forces to be considered as steady-state (Bird et al. 1987). Besides the bursting strength, Fig. 2 reveals that after the cell bursts (around point C), the force does not immediately return to zero (FG). ']?he residual resistance might possibly be due to the cell debris between the probes which can be observed microscopically. The strength of h y b r i d o m a cells from the synchronous culture was measured. It was expected that cells of a given size taken at a particular time from such a culture would have similar bursting strengths. Multiple measurements on such cells would test the validity of the method. Six cells actually chosen were of diameters l l . l _ 0 . 4 1 x m . All cells were selected, captured and squeezed within a total of 45 min. The strengths were 2.4, 2.1, 2.5, 2.4, 2.6, 2.1 ixN respectively. The m e a n strength was 2.4 txN and the m e a n squared deviation was 0.21 txN. The result clearly demonstrates the reproducibility of the technique. Figure 3 shows strength versus the size of h y b r i d o m a cells taken from the continuous culture (after steadystate was established). Cells from any particular sample were tested within ,45 min of sampling. Results were collated from ten different samples within 10 days. As might be expected, the strength of the cells depended on cell size. Interpretation of cell strength in terms of cell m e m b r a n e properties is possible and will be reported elsewhere. At any given size, the strength variations shown in Fig. 3 suggested that cells from continuous culture are less homogeneous than those from synchronous culture. The distribution of strengths in cell populations is to be studied as part of future work

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Acknowledgements. This research was supported by the Science and Engineering Research Council, UK. Dr. N Bevoric is thanked for discussions on micromanipulation. Dr. M A1-Rubeai and Mr. D Jan are thanked for providing cell samples.

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o n the d e p e n d e n c e o f cell strength o n culture conditions. T h e results s h o w that the t e c h n i q u e described here is suitable f o r s t u d y i n g the bursting strength o f m a m m a l ian cells. It will permit direct c o m p a r i s o n s o f different cell lines, a n d studies o n the effect o f culture conditions on cell m e c h a n i c a l properties. It provides a tool f o r investigating h o w such properties d e p e n d on cell structure a n d physiology. This i n f o r m a t i o n is essential if cell strength is to be m a n i p u l a t e d a n d controlled, a n d if

A1-Rubeai M, Emery AN (1990) Mechanisms and kinetics of monoclonal antibody synthesis and secretion in synchronous and asynchronous hybridoma cell cultures. J Biotechnol 16:67-86 Bird RB, Armstrong RC, Hassager O (1987) Dynamics of polymeric liquids, vol. 1, appendix B. Wiley, New York Bleim R (1989) A need for systematic investigations into the material properties of cultured animal cells. Trends Biotechnol 7:197-200 Cherry R_S, Kwon K (1990) Transient shear stresses on a suspension cell in turbulence. Biotechnol Bioeng 36:563-571 Evans EA, Skalak R (1980) Mechanics and thermodynamics of biomembranes. CRC Press, Boca Raton, pp 148-155 Hochmuth RM (1987) Properties of red blood cells. In: Skalak R, Chien S (eds) Handbook of bioengineering. McGraw-Hill, New York, pp 12.1-12.17 Petersen JF, Mclntire LV, Papoutsakis ET (1988) Shear sensitivity of cultured hybridoma cells (CRL-8018) depends on mode of growth, culture age and metabolite concentration. J Biotechnol 7:229-246 Sato M, Levesque MJ, Nerem RM (1987) An application of the micropipette technique to the measurement of the mechanical properties of cultured bovine aortic endothelial cells. J Biomech Eng 109:27-34 Smith CG, Greenfield PF, Randerson DH (1987) A technique for determining the shear sensitivity of mammalian cells in suspension culture. Biotechnol Tech 1:39-44 Stathopoulos NA, Hellums JD (1985) Shear stress effects on human embryonic kidney cells in vitro. Biotechnol Bioeng 36:563-571

A novel micromanipulation technique for measuring the bursting strength of single mammalian cells.

Information about the bursting strength of animal cells is essential if the mechanisms of cell damage in bioreactors are to be understood, and if cell...
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