0 1991 Wiley-Liss, Inc.
Cytometry 12505-510 (1991)
Laser Induced Cell Fusion in Combination With Optical Tweezers: The Laser Cell Fusion Trap Rosemarie Wiegand Steubing, Steve Cheng, William H. Wright, Yasuyuki Numajiri, and Michael W. Berns Beckman Laser Institute and Medical Clinic, University of California, Irvine, Irvine, California 92715 Received for publication February 1, 1990; accepted May 17, 1990
A single-beam gradient force optical trap was combined with a pulsed UV laser microbeam in order to perform laser induced cell fusion. This combination offers the possibility to selectively fuse two single cells without critical chemical or electrical treatment. The optical trap was created by directing a NdYAG laser, at a wavelength of 1.06 pm, into a microscope and focusing the laser beam with a high numerical aperture objective. The UV laser microbeam, produced by a nitrogenpumped dye laser (366 nm),was collinear
At present, a number of different fusion techniques are available to introduce genetic material and molecules into cells or to produce hybrid cells with various properties such as the production of monoclonal antibodies. Polyethylene glycol (PEG) induced cell-cell fusion has become a standard technique (81, especially in the hybridoma technology. PEG induced fusions are easy to perform, and a high number of cells can be fused in a short time. One drawback, however, is its low fusion efficiency and the fact that many unwanted fusion products are created. With the introduction of cell fusion by inhomogeneous electrical fields in 1980 (11,211, the fusion efficiencies under controlled conditions could be improved dramatically. Laser-induced cell fusion was first used to fuse two cells which were already touching each other. The embryonic cells used in these experiments (14) were surrounded by a transparent but otherwise unpenetrable egg shell; therefore, the laser microbeam provided a unique way to perform the fusion. Later on, this selective fusion technique (15) was also used to fuse B lymphocytes with myeloma cells in suspension and to fuse plant protoplasts with each other (18,19). The mammalian cells, however, could only be fused by a UV laser microbeam after binding them together via a n avidinbiotin bridge (10). Although the avidin-biotin bridge created a very specific bond between selected cells, it limited the fusion yield and it was a time consuming
with the trapping beam. Once inside the trap, two cells could be fused with several pulses of the UV laser microbeam, attenuatedto an energy of 1 pJ/pulse in the object plane. This method of laser induced cell fusion should provide increased selectivity and efficiency in generating viable hybrid cells.
-
Key terms: Optical trapping, cell micromanipulation, laser microbeam, hybrid cell
process. A novel approach, described in this paper, is the use of a n optical trap to establish the necessary contact between two cells. Once inside the trap, two cells were fused using a pulsed laser at a wavelength of 366 nm. The combination of a n optical trap with a pulsed UV laser (laser cell fusion trap) offers a versatile way to perform selective cell fusions at the single cell level which does not depend on any natural cell contact or specific cell receptors. I n this study, we employed a single beam optical trap similar to that described by Ashkin et al. (1,2). Biological particles such as Exoli, lymphocytes, red blood cells, and macrophages could be trapped successfully, introducing the “optical tweezer” into the field of biological micromanipulation (3-7,17). The physical principles of optical trapping have been described in detail (1,2). Briefly, the cell is pulled laterally into the center of the beam due to the transverse intensity gradient of the gaussian laser beam. For a spot diameter on the order of the wavelength of light, the force due to the axial laser beam gradient pointing towards the beam waist is greater than the scattering force in the direction of the light. This pulls the cell vertically to a point just below the laser beam waist, thereby completing the 3-D trap. If the trapping beam is not focused to a very small spot, the cell will be pushed in the direction of the beam. Cells can still be “trapped” however by pushing them against the microscope slide and drag-
STEUBING ET AL.
506 h = 3 6 6 nm
h
1.06 prn
FIG.1. Schematic diagram of two cells in the laser fusion trap. The transverse force denoted as F, is due to the intensity gradient of the trapping infrared laser beam. The axial force denoted as Fa points in the direction of the laser beam. The optical trap is used to drag the cell towards the second cell. Once the close contact is established, the cells are fused by the UV laser microbeam.
ging them laterally with the laser beam. This was the case with our system. Figure 1 shows the geometry of the two cells in the laser fusion trap. As indicated by the arrows, the transverse force F, pulls one cell towards the other, while the axial force Fa keeps the trapped cell on the surface of the slide, along which the cell is moved. As soon a s both cells are in close contact, they can be fused with the UV laser microbeam. Note that the optical trap was used to move one cell adjacent to a second cell already stuck to the slide. In the following, we describe a new instrument which combines a n optical trap laser system with a pulsed UV laser to perform cell fusion. This unique method permits the selective fusion of any two cells in suspension, not just those that are in close contact only by chance. Parameters such as the number of pulses and energies required for laser fusion, the incubation temperature of the cells during the fusion process, and the effect of PEG on the fusion frequency are presented.
MATERIALS AND METHODS Instrumentation The instrumental setup for the laser fusion trap is shown in Figure 2. A continuous wave Nd:YAG laser (Quantronix model 116, Smithtown, NY) emitting at a wavelength of 1.06 pm was used to create the optical trap. The infrared laser beam passes through a n attenuator, a polarizer, and a dichroic mirror before being directed into the microscope via the dichroic reflector. The nitrogen laser pumped dye laser (Photon Technologies International, model PL2300 and PL201, respectively) is focused by a 15 cm focusing lens to decrease the divergence of the dye laser beam, and then reflected to the beamsplitter, which combines the two laser
beams. The dichroic reflector reflects both the IR beam and the UV beam into the microscope while allowing the visible light to pass to the video camera. A focusing lens was used to fill the back aperture of the objective lens to achieve a small spot size a t the trap. The objective was a water immersion type, with a numerical aperture of 1.2 and a magnification of 63 x , so that the experiments could be performed without a coverslip. The spot diameter of the focused infrared trapping beam is estimated to be = 1.5 pm. All experiments were recorded on videotape and later analyzed using a n image processor.
Power Measurements Nd:YAG laser. The power of the Nd:YAG laser was monitored before the beam entered the microscope using a Scientech model 362 meter (Boulder, CO). The meter was located before the beam splitter, as indicated in Figure 2. It was calibrated against a second meter (Scientech 362) that measured the power transmitted through the objective. The power a t the objective for typical trap conditions during fusion experiments was in the range of 220 mW. Dye laser. The output energy of the dye laser was measured using a pyroelectric detector (Gentec, ED100A, Quebec, Canada) connected to a n oscilloscope. An energy of 60 pJ was measured after the beam passed through the first focusing lens. The energy decreased to approximately 1 pJ/pulse after the beam passed through the objective. Preparation of Cells The myeloma cell line NS-1 (American Type Culture Collection, number TIB18) used for the experiments was cultured in RPMI 1640 (Hybrimax, Sigma Chemical Co., St. Louis, MO) supplemented with 10% fetal bovine serum (FBS), 1%glutamine (200 mM), and 1% penicillin (10,000 units/ml) and streptomycin (10,000 mg/ml). Prior to the fusion experiments, the cells were centrifuged for 10 min a t 250g and resuspended in tyrosine and tryptophan deficient RPMI medium (tyd trp-RPMI). This medium has been used because it has been reported that tyrosine and tryptophan in combination with riboflavin produce toxic photoproducts upon UV irradiation (16). A 35 mm diameter Petri dish (Falcon; not tissue culture treated) was fitted with a glass slide imprinted with a 2-D grid (Cel-Line Assn. Newfield, NJ), cut into 1.0 cm x 2.0 cm pieces. The Petri dish containing the slide was filled with approximately 2 ml of tyr/trp- RPMI. An aliquot of the resuspended NS-1 cells was drawn u p into a microcapillary tube and plated dropwise into each of the center squares (area = 1 mm2) of the hydrophobic grid. For the determination of the fusion frequency in medium containing polyethylene glycol (PEG), we used PEG with a molecular weight of 3,000-3,700 (Sigma Chemical Co., St. Louis, MO), which was adjusted to a
THE LASER CELL FUSION TRAP
507
1
Camera
Imaging lens
Filter
1
Dichroic reflector
Focusq lens 2
Monitor
Objectivc lens
L
meter
Chamber
106um Nd YAG laser
FIG.2. Simplified diagram of the combination of the single beam gradient trap with the UV laser microbeam. A detailed description is given in the text.
50% solution in Hank’s Balanced Salt Solution (HBSS), and then diluted to 1% and 0.1% using RPMI medium. Immediately after the fusions, the manipulation chamber was transferred to a n inverted microscope to isolate the fused cells with a microcapillary. The fused cells were removed from the manipulation chamber and each one plated into one well of a 96-well plate. Each well contained 100 ~1 of a mixture of NS-1 conditioned medium and RPMI medium in a 1:4 ratio, supplemented with 10-20% FBS, 1%glutamine, 1% penicillin/streptomycin, and 1% Oxaloacetate-Pyruvate-Insulin (OPI) (9).
RESULTS Homokaryon Production (NS-1 cells) and Fusion Frequency A typical laser-induced cell fusion is illustrated in Figure 3. The cells were prepared and placed into the manipulation chamber a s described above. The chamber was located under the microscope and kept a t approximately 37°C using a temperature controlled sample stage. Figure 3a shows two cells which were brought near each other by the optical trap but are not
yet touching. The lower cell was then moved very close to the upper cell, as shown in Figure 3b. The dye laser was then targeted to hit between the two cells. A few pulses of the dye laser were sufficient to induce fusion, indicated in Figure 3c. In this particular example it took approximately 5 min until the cells rounded up, as shown in Figure 3d. The pulse rate of the dye laser was adjusted between 6 and 15 Hz to produce optimal fusion conditions. This repetition rate has been chosen because it is known from previous experiments (20) that a low energy of about 1 pJ/pulse and a repetition rate a t about 10 Hz permits fusion without destroying the cells. The repetition rate and the energy density are indirectly proportional to each other; that is, fewer pulses are required when higher energetic pulses are used. This has been shown in cell killing experiments (unpublished data). Depending on the pair of cells to be fused, 2 to 50 pulses were required. In all fusion experiments, it took from 2 to 8 min until the fused cells rounded up and became one hybrid cell. Occasionally, if a pair of cells did not continue rounding up to a hybrid cell after the first set of pulses, a second set of pulses were applied.
508
STEUBING ET AL.
FIG.3. Laser-induced cell fusion of one pair of cells in the optical trap. A: The lower one of the two cells has been brought near the upper cell by dragging it using the optical trap. Note that the cells are not in contact yet. B: Close cell contact is provided now after dragging the lower cell in the trap very close to the other cell. The UV laser
beam was turned on at this moment. C: After being hit by approximately ten pulses of the 366 nm laser microbeam, the cells started to fuse. Note the separating plasma membrane disappearing, D About 5 min post-fusion. The hybrid cell has rounded up.
The fusion frequency of laser induced cell fusion for this method was a function of two parameters: the temperature and the presence of PEG. As indicated in Table 1, the fusion rate in the culture medium at room temperature was 1.3 1.0%. When fusions were performed in the same medium and a t room temperature, but with the addition of 1%PEG, the fusion frequency could be increased to 8.6 t 3.2%. When the cells were kept a t 37°C during the fusion process, a similar frequency of 11 s 4.0% was obtained. Note, that in the latter case the concentration of the PEG was only 0.1%,
indicating that the temperature is a n important parameter.
*
DISCUSSION The preliminary results from our laser fusion trap clearly indicate that highly selective cell fusions can be performed with cells in suspension. We determined the fusion frequency under various conditions to compare this technique to other cell fusion methods. It is superior in that unwanted fusion products are not produced since only preselected pairs of cells are fused. One can
509
THE LASER CELL FUSION TRAP
Table 1 Fusion Freauencies of Laser-Induced Cell Fusion
In culture medium“ In culture medium containing 1%PEG In culture medium containing 0.1% PEG at 37°C
Number of attemots 75
Number of fusions 1
112
9
54
6
Fusion frequency (%)”
1.3 ? 1.0 8.6
+ 3.2
11.0 t 4.0
“The data are the mean ? SD of three independent experiments. “The culture medium used during the irradiations was tyri trp- RPMI without FBS.
precisely trap selected cells, if a n appropriate label is available. For example, if one cell can be labeled with a fluorescent molecule such a s a n anti-receptor antibody or any fluorescent molecule t h a t is bound on the surface of the particular cell, its selection in the laser fusion trap using fluorescence microscopy is possible. Fusions of such preselected pairs of cells have been proved in (201, where the contact between the cells was established chemically. Laser-induced cell fusion using cells preselected by fluorescence techniques and manipulated by the optical trap will be more flexible and less time consuming because no such chemical binding is necessary. Furthermore, this method may be amenable to automation. An example is the instrument described by Buican et al. (6,7), using a computer-controlled optical trap system for cell manipulation and robotics. The following parameters have been established, or even improved, over the experiments conducted so far in this area (14,15,18,19). First, the introduction of the “optical tweezer” will broaden the application of laserinduced cell fusion in biotechnology because the cells are brought together immediately before the fusions. Second, the addition of 1% PEG to the fusion medium dramatically enhances the laser induced fusion frequency. It is reasonable to believe that other agents with the capability to aggregate cells might be effective, too. Although the influence of PEG on the fusion of cells is not clear, a direct interaction with membrane constituents has been ruled out. Apparently, PEG alters the physicochemical properties of the water surrounding the cells, thereby creating a depletion layer between the two opposing membranes (13). PEG a t such low concentrations used in our experiments, however, does not possess any fusing activity per se. Currrently, we have been unsuccessful in obtaining a cloned cell hybrid following laser fusion. Therefore, the viability of the fused cells has not been demonstrated. Since it has been shown by Schierenberg et al. (14) that fusion products of embryonic cells were able to divide, we think that the failure to see the division of the created hybrids was due to cell handling after the fusion and the small numbers of fused cells followed. To
date, the exact number of successful fusions necessary to isolate one viable clone is not known. Peters et al. (12) calculated that approximately 600 fusion products are necessary to isolate 1-3 viable hybrid cells. This statistical value, however, is not related to the actual number of successful fusions. The laser fusion trap should allow precise determination of the fusion frequency and efficiency.
CONCLUSION The combination of a n optical trap and a pulsed UV laser was used for very selective and precise fusions of two preselected cells in suspension. The two cells were selected by simply dragging one cell toward a second cell with the aid of the optical trap. This novel approach also provides a new and promising application of optical trapping in biotechnology. The fusion frequency can be increased from 1% to 8%when 1% PEG is added to the fusion medium at room temperature. Keeping the cells a t = 37°C during the fusion experiments resulted in a fusion frequency of 11%.Observation of the success of each fusion indicated that the fused cells are viable for a t least the first 2-3 h post fusion. The ability of the hybrid cells to divide following fusion in our laser fusion trap has not yet been shown. Ultimately, our laser fusion trap may be very useful for highly selective cell fusions where only limited amounts of cells are available. ACKNOWLEDGMENTS This work was supported by grants from the NIH (RR01192), SDIO (84-88-C-0025), and the Beckman Laser Institute Foundation. We would like to thank Chung-Ho Sun, Ph.D., Bruce J. Tromberg, Ph.D. (both from Beckman Laser Institute), Karin Schutze, Ph.D. (Institute for Zoology, University of California, Berkeley), and Tudor N. Buican, Ph.D. (Los Alamos National Laboratory, NM) for helpful and encouraging discussions. LITERATURE CITED 1. Ashkin A: Trapping of atoms by resonance radiation pressure. Phys Rev Lett 40:729, 1978. 2. Ashkin A, Dziedzic JM, Bjorkholm J , Chu S: Observation of a single-beam gradient force optical trap for dielectric particles. Opt Lett 11:288-290, 1986. 3. Ashkin A, Dziedzic JM, Yamane T: Optical trapping and manipulation of viruses and bacteria. Science 2351517-1520, 1987. 4. Ashkin A, Dziedzic JM, Yamane T: Optical trapping and manipulation of single cells using infrared laser beams. Nature 330: 769-771, 1987. 5. Berns MW, Wright WH, Tromberg BJ, Profeta GA, Andrews JJ, Walter RJ: Use of a laser-induced optical force trap to study chromosome movement on the mitotic spindle. Proc Natl Acad Sci USA 86:4539-4543, 1989. 6. Buican TN, Smyth MJ, Crissman HA, Salzman GC, Stewart CC, Martin JC: Automated single-cell manipulation and sorting by light trapping. Appl Opt 26:5311-5316, 1987. 7. Buican TN, Neagley DL, Morrison WL, Upham BW: Optical trapping, cell manipulation and robotics. In: New Technologies in Cytometry. SPIE Vol. 1063, 1989, pp 190-197. 8. Fazekas de St. Groth S, Scheidegger D: Production of monoclonal
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9. 10. 11. 12. 13. 14. 15.
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antibodies: Strategy and tactics. J Immunol Methods 35:l-21, 1980. Harlow E, Lane D: Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1988, p 200. Lo M, Tsong T, Conrad M, Strittmatter S, Hester L, Snyder S: Monoclonal antibody production hy receptor-mediated electrically induced cell fusion. Nature 310:792-794, 1984. Neumann E, Gerisch G, Opatz K: Cell fusion induced by high electric impulses applied t o Dictyostelium. Naturwissenschaften 67:414-415. 1980. Peters J , Baumgarten H, Schultze M: Monoklonale Antikorper. Herstellung und Charakterisierung. Springer, Berlin, 1985. Pratsch L, Herrmann A, Ilona A, Meyer HW: The influence of poly(ethy1ene glycol) on the molecular dynamics within the Glycocalyx. Biochim Biophys Acta 980:146-154, 1989. Schierenherg E: Altered cell division rates after laser-induced cell fusion in nematode embryos. Dev Biol 101:240-245, 1984. Schierenberg E: Laser-induced cell fusion. In: Cell Fusion, Sowers AE (ed). Plenum Press, New York, 1987, pp 409-418.
16. Stoien JD, Wang FLJ: Effect of near-ultraviolet and visible light on mammalian cells in culture. 11. Formation of toxic photoproducts in tissue culture medium by blacklight. Proc Natl Acad Sci USA 71:3961-3965, 1974. 17. Tadir Y, Wright WH, Vafa 0, Ord T, Asch R, Berns MW: Micromanipulation of sperm by a laser generated optical trap. Fertil Steril 52:870-873, 1989. 18. Wiegand Steubing R, Zimmermann K, Monajembashi S, Schafer H, Hansch GM, Greulich K, Wolfrum J: Laser induced fusion of myeloma cells and B-lymphocytes. Immunobiology 173:320, 1986 (Erratum Immunobiology 174:120, 1987). 19. Wiegand-Steubing R, Weber G, Zimmermann K, Monajembashi S, Greulich K, Wolfrum J: Laser induced fusion of mammalian cells and plant protoplasts. J Cell Science 88:145-149, 1987. 20. Wiegand R: EinfluR von chemisch und laser-induzierten Membranporen auf das Verhalten und die Fusion kernhaltiger Zellen, Ph.D. thesis, University of Heidelberg, West Germany, 1988. 21. Zimmermann U: Electric field-mediated fusion and related electrical phenomena. Biochem Biophys Acta 694:227-277, 1982.
Cytometry 12505-510 (1991)
Laser Induced Cell Fusion in Combination With Optical Tweezers: The Laser Cell Fusion Trap Rosemarie Wiegand Steubing, Steve Cheng, William H. Wright, Yasuyuki Numajiri, and Michael W. Berns Beckman Laser Institute and Medical Clinic, University of California, Irvine, Irvine, California 92715 Received for publication February 1, 1990; accepted May 17, 1990
A single-beam gradient force optical trap was combined with a pulsed UV laser microbeam in order to perform laser induced cell fusion. This combination offers the possibility to selectively fuse two single cells without critical chemical or electrical treatment. The optical trap was created by directing a NdYAG laser, at a wavelength of 1.06 pm, into a microscope and focusing the laser beam with a high numerical aperture objective. The UV laser microbeam, produced by a nitrogenpumped dye laser (366 nm),was collinear
At present, a number of different fusion techniques are available to introduce genetic material and molecules into cells or to produce hybrid cells with various properties such as the production of monoclonal antibodies. Polyethylene glycol (PEG) induced cell-cell fusion has become a standard technique (81, especially in the hybridoma technology. PEG induced fusions are easy to perform, and a high number of cells can be fused in a short time. One drawback, however, is its low fusion efficiency and the fact that many unwanted fusion products are created. With the introduction of cell fusion by inhomogeneous electrical fields in 1980 (11,211, the fusion efficiencies under controlled conditions could be improved dramatically. Laser-induced cell fusion was first used to fuse two cells which were already touching each other. The embryonic cells used in these experiments (14) were surrounded by a transparent but otherwise unpenetrable egg shell; therefore, the laser microbeam provided a unique way to perform the fusion. Later on, this selective fusion technique (15) was also used to fuse B lymphocytes with myeloma cells in suspension and to fuse plant protoplasts with each other (18,19). The mammalian cells, however, could only be fused by a UV laser microbeam after binding them together via a n avidinbiotin bridge (10). Although the avidin-biotin bridge created a very specific bond between selected cells, it limited the fusion yield and it was a time consuming
with the trapping beam. Once inside the trap, two cells could be fused with several pulses of the UV laser microbeam, attenuatedto an energy of 1 pJ/pulse in the object plane. This method of laser induced cell fusion should provide increased selectivity and efficiency in generating viable hybrid cells.
-
Key terms: Optical trapping, cell micromanipulation, laser microbeam, hybrid cell
process. A novel approach, described in this paper, is the use of a n optical trap to establish the necessary contact between two cells. Once inside the trap, two cells were fused using a pulsed laser at a wavelength of 366 nm. The combination of a n optical trap with a pulsed UV laser (laser cell fusion trap) offers a versatile way to perform selective cell fusions at the single cell level which does not depend on any natural cell contact or specific cell receptors. I n this study, we employed a single beam optical trap similar to that described by Ashkin et al. (1,2). Biological particles such as Exoli, lymphocytes, red blood cells, and macrophages could be trapped successfully, introducing the “optical tweezer” into the field of biological micromanipulation (3-7,17). The physical principles of optical trapping have been described in detail (1,2). Briefly, the cell is pulled laterally into the center of the beam due to the transverse intensity gradient of the gaussian laser beam. For a spot diameter on the order of the wavelength of light, the force due to the axial laser beam gradient pointing towards the beam waist is greater than the scattering force in the direction of the light. This pulls the cell vertically to a point just below the laser beam waist, thereby completing the 3-D trap. If the trapping beam is not focused to a very small spot, the cell will be pushed in the direction of the beam. Cells can still be “trapped” however by pushing them against the microscope slide and drag-
STEUBING ET AL.
506 h = 3 6 6 nm
h
1.06 prn
FIG.1. Schematic diagram of two cells in the laser fusion trap. The transverse force denoted as F, is due to the intensity gradient of the trapping infrared laser beam. The axial force denoted as Fa points in the direction of the laser beam. The optical trap is used to drag the cell towards the second cell. Once the close contact is established, the cells are fused by the UV laser microbeam.
ging them laterally with the laser beam. This was the case with our system. Figure 1 shows the geometry of the two cells in the laser fusion trap. As indicated by the arrows, the transverse force F, pulls one cell towards the other, while the axial force Fa keeps the trapped cell on the surface of the slide, along which the cell is moved. As soon a s both cells are in close contact, they can be fused with the UV laser microbeam. Note that the optical trap was used to move one cell adjacent to a second cell already stuck to the slide. In the following, we describe a new instrument which combines a n optical trap laser system with a pulsed UV laser to perform cell fusion. This unique method permits the selective fusion of any two cells in suspension, not just those that are in close contact only by chance. Parameters such as the number of pulses and energies required for laser fusion, the incubation temperature of the cells during the fusion process, and the effect of PEG on the fusion frequency are presented.
MATERIALS AND METHODS Instrumentation The instrumental setup for the laser fusion trap is shown in Figure 2. A continuous wave Nd:YAG laser (Quantronix model 116, Smithtown, NY) emitting at a wavelength of 1.06 pm was used to create the optical trap. The infrared laser beam passes through a n attenuator, a polarizer, and a dichroic mirror before being directed into the microscope via the dichroic reflector. The nitrogen laser pumped dye laser (Photon Technologies International, model PL2300 and PL201, respectively) is focused by a 15 cm focusing lens to decrease the divergence of the dye laser beam, and then reflected to the beamsplitter, which combines the two laser
beams. The dichroic reflector reflects both the IR beam and the UV beam into the microscope while allowing the visible light to pass to the video camera. A focusing lens was used to fill the back aperture of the objective lens to achieve a small spot size a t the trap. The objective was a water immersion type, with a numerical aperture of 1.2 and a magnification of 63 x , so that the experiments could be performed without a coverslip. The spot diameter of the focused infrared trapping beam is estimated to be = 1.5 pm. All experiments were recorded on videotape and later analyzed using a n image processor.
Power Measurements Nd:YAG laser. The power of the Nd:YAG laser was monitored before the beam entered the microscope using a Scientech model 362 meter (Boulder, CO). The meter was located before the beam splitter, as indicated in Figure 2. It was calibrated against a second meter (Scientech 362) that measured the power transmitted through the objective. The power a t the objective for typical trap conditions during fusion experiments was in the range of 220 mW. Dye laser. The output energy of the dye laser was measured using a pyroelectric detector (Gentec, ED100A, Quebec, Canada) connected to a n oscilloscope. An energy of 60 pJ was measured after the beam passed through the first focusing lens. The energy decreased to approximately 1 pJ/pulse after the beam passed through the objective. Preparation of Cells The myeloma cell line NS-1 (American Type Culture Collection, number TIB18) used for the experiments was cultured in RPMI 1640 (Hybrimax, Sigma Chemical Co., St. Louis, MO) supplemented with 10% fetal bovine serum (FBS), 1%glutamine (200 mM), and 1% penicillin (10,000 units/ml) and streptomycin (10,000 mg/ml). Prior to the fusion experiments, the cells were centrifuged for 10 min a t 250g and resuspended in tyrosine and tryptophan deficient RPMI medium (tyd trp-RPMI). This medium has been used because it has been reported that tyrosine and tryptophan in combination with riboflavin produce toxic photoproducts upon UV irradiation (16). A 35 mm diameter Petri dish (Falcon; not tissue culture treated) was fitted with a glass slide imprinted with a 2-D grid (Cel-Line Assn. Newfield, NJ), cut into 1.0 cm x 2.0 cm pieces. The Petri dish containing the slide was filled with approximately 2 ml of tyr/trp- RPMI. An aliquot of the resuspended NS-1 cells was drawn u p into a microcapillary tube and plated dropwise into each of the center squares (area = 1 mm2) of the hydrophobic grid. For the determination of the fusion frequency in medium containing polyethylene glycol (PEG), we used PEG with a molecular weight of 3,000-3,700 (Sigma Chemical Co., St. Louis, MO), which was adjusted to a
THE LASER CELL FUSION TRAP
507
1
Camera
Imaging lens
Filter
1
Dichroic reflector
Focusq lens 2
Monitor
Objectivc lens
L
meter
Chamber
106um Nd YAG laser
FIG.2. Simplified diagram of the combination of the single beam gradient trap with the UV laser microbeam. A detailed description is given in the text.
50% solution in Hank’s Balanced Salt Solution (HBSS), and then diluted to 1% and 0.1% using RPMI medium. Immediately after the fusions, the manipulation chamber was transferred to a n inverted microscope to isolate the fused cells with a microcapillary. The fused cells were removed from the manipulation chamber and each one plated into one well of a 96-well plate. Each well contained 100 ~1 of a mixture of NS-1 conditioned medium and RPMI medium in a 1:4 ratio, supplemented with 10-20% FBS, 1%glutamine, 1% penicillin/streptomycin, and 1% Oxaloacetate-Pyruvate-Insulin (OPI) (9).
RESULTS Homokaryon Production (NS-1 cells) and Fusion Frequency A typical laser-induced cell fusion is illustrated in Figure 3. The cells were prepared and placed into the manipulation chamber a s described above. The chamber was located under the microscope and kept a t approximately 37°C using a temperature controlled sample stage. Figure 3a shows two cells which were brought near each other by the optical trap but are not
yet touching. The lower cell was then moved very close to the upper cell, as shown in Figure 3b. The dye laser was then targeted to hit between the two cells. A few pulses of the dye laser were sufficient to induce fusion, indicated in Figure 3c. In this particular example it took approximately 5 min until the cells rounded up, as shown in Figure 3d. The pulse rate of the dye laser was adjusted between 6 and 15 Hz to produce optimal fusion conditions. This repetition rate has been chosen because it is known from previous experiments (20) that a low energy of about 1 pJ/pulse and a repetition rate a t about 10 Hz permits fusion without destroying the cells. The repetition rate and the energy density are indirectly proportional to each other; that is, fewer pulses are required when higher energetic pulses are used. This has been shown in cell killing experiments (unpublished data). Depending on the pair of cells to be fused, 2 to 50 pulses were required. In all fusion experiments, it took from 2 to 8 min until the fused cells rounded up and became one hybrid cell. Occasionally, if a pair of cells did not continue rounding up to a hybrid cell after the first set of pulses, a second set of pulses were applied.
508
STEUBING ET AL.
FIG.3. Laser-induced cell fusion of one pair of cells in the optical trap. A: The lower one of the two cells has been brought near the upper cell by dragging it using the optical trap. Note that the cells are not in contact yet. B: Close cell contact is provided now after dragging the lower cell in the trap very close to the other cell. The UV laser
beam was turned on at this moment. C: After being hit by approximately ten pulses of the 366 nm laser microbeam, the cells started to fuse. Note the separating plasma membrane disappearing, D About 5 min post-fusion. The hybrid cell has rounded up.
The fusion frequency of laser induced cell fusion for this method was a function of two parameters: the temperature and the presence of PEG. As indicated in Table 1, the fusion rate in the culture medium at room temperature was 1.3 1.0%. When fusions were performed in the same medium and a t room temperature, but with the addition of 1%PEG, the fusion frequency could be increased to 8.6 t 3.2%. When the cells were kept a t 37°C during the fusion process, a similar frequency of 11 s 4.0% was obtained. Note, that in the latter case the concentration of the PEG was only 0.1%,
indicating that the temperature is a n important parameter.
*
DISCUSSION The preliminary results from our laser fusion trap clearly indicate that highly selective cell fusions can be performed with cells in suspension. We determined the fusion frequency under various conditions to compare this technique to other cell fusion methods. It is superior in that unwanted fusion products are not produced since only preselected pairs of cells are fused. One can
509
THE LASER CELL FUSION TRAP
Table 1 Fusion Freauencies of Laser-Induced Cell Fusion
In culture medium“ In culture medium containing 1%PEG In culture medium containing 0.1% PEG at 37°C
Number of attemots 75
Number of fusions 1
112
9
54
6
Fusion frequency (%)”
1.3 ? 1.0 8.6
+ 3.2
11.0 t 4.0
“The data are the mean ? SD of three independent experiments. “The culture medium used during the irradiations was tyri trp- RPMI without FBS.
precisely trap selected cells, if a n appropriate label is available. For example, if one cell can be labeled with a fluorescent molecule such a s a n anti-receptor antibody or any fluorescent molecule t h a t is bound on the surface of the particular cell, its selection in the laser fusion trap using fluorescence microscopy is possible. Fusions of such preselected pairs of cells have been proved in (201, where the contact between the cells was established chemically. Laser-induced cell fusion using cells preselected by fluorescence techniques and manipulated by the optical trap will be more flexible and less time consuming because no such chemical binding is necessary. Furthermore, this method may be amenable to automation. An example is the instrument described by Buican et al. (6,7), using a computer-controlled optical trap system for cell manipulation and robotics. The following parameters have been established, or even improved, over the experiments conducted so far in this area (14,15,18,19). First, the introduction of the “optical tweezer” will broaden the application of laserinduced cell fusion in biotechnology because the cells are brought together immediately before the fusions. Second, the addition of 1% PEG to the fusion medium dramatically enhances the laser induced fusion frequency. It is reasonable to believe that other agents with the capability to aggregate cells might be effective, too. Although the influence of PEG on the fusion of cells is not clear, a direct interaction with membrane constituents has been ruled out. Apparently, PEG alters the physicochemical properties of the water surrounding the cells, thereby creating a depletion layer between the two opposing membranes (13). PEG a t such low concentrations used in our experiments, however, does not possess any fusing activity per se. Currrently, we have been unsuccessful in obtaining a cloned cell hybrid following laser fusion. Therefore, the viability of the fused cells has not been demonstrated. Since it has been shown by Schierenberg et al. (14) that fusion products of embryonic cells were able to divide, we think that the failure to see the division of the created hybrids was due to cell handling after the fusion and the small numbers of fused cells followed. To
date, the exact number of successful fusions necessary to isolate one viable clone is not known. Peters et al. (12) calculated that approximately 600 fusion products are necessary to isolate 1-3 viable hybrid cells. This statistical value, however, is not related to the actual number of successful fusions. The laser fusion trap should allow precise determination of the fusion frequency and efficiency.
CONCLUSION The combination of a n optical trap and a pulsed UV laser was used for very selective and precise fusions of two preselected cells in suspension. The two cells were selected by simply dragging one cell toward a second cell with the aid of the optical trap. This novel approach also provides a new and promising application of optical trapping in biotechnology. The fusion frequency can be increased from 1% to 8%when 1% PEG is added to the fusion medium at room temperature. Keeping the cells a t = 37°C during the fusion experiments resulted in a fusion frequency of 11%.Observation of the success of each fusion indicated that the fused cells are viable for a t least the first 2-3 h post fusion. The ability of the hybrid cells to divide following fusion in our laser fusion trap has not yet been shown. Ultimately, our laser fusion trap may be very useful for highly selective cell fusions where only limited amounts of cells are available. ACKNOWLEDGMENTS This work was supported by grants from the NIH (RR01192), SDIO (84-88-C-0025), and the Beckman Laser Institute Foundation. We would like to thank Chung-Ho Sun, Ph.D., Bruce J. Tromberg, Ph.D. (both from Beckman Laser Institute), Karin Schutze, Ph.D. (Institute for Zoology, University of California, Berkeley), and Tudor N. Buican, Ph.D. (Los Alamos National Laboratory, NM) for helpful and encouraging discussions. LITERATURE CITED 1. Ashkin A: Trapping of atoms by resonance radiation pressure. Phys Rev Lett 40:729, 1978. 2. Ashkin A, Dziedzic JM, Bjorkholm J , Chu S: Observation of a single-beam gradient force optical trap for dielectric particles. Opt Lett 11:288-290, 1986. 3. Ashkin A, Dziedzic JM, Yamane T: Optical trapping and manipulation of viruses and bacteria. Science 2351517-1520, 1987. 4. Ashkin A, Dziedzic JM, Yamane T: Optical trapping and manipulation of single cells using infrared laser beams. Nature 330: 769-771, 1987. 5. Berns MW, Wright WH, Tromberg BJ, Profeta GA, Andrews JJ, Walter RJ: Use of a laser-induced optical force trap to study chromosome movement on the mitotic spindle. Proc Natl Acad Sci USA 86:4539-4543, 1989. 6. Buican TN, Smyth MJ, Crissman HA, Salzman GC, Stewart CC, Martin JC: Automated single-cell manipulation and sorting by light trapping. Appl Opt 26:5311-5316, 1987. 7. Buican TN, Neagley DL, Morrison WL, Upham BW: Optical trapping, cell manipulation and robotics. In: New Technologies in Cytometry. SPIE Vol. 1063, 1989, pp 190-197. 8. Fazekas de St. Groth S, Scheidegger D: Production of monoclonal
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