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Resolution of cells by centrifugal elutriation
ANALYIICAI
98.
HIOCHthllSlRY
112-I
Resolution
15 (1979)
of Cells by Centrifugal
WILSON D. GRANT AND MARTIN
Received
Cell and can
February
separation using the Beckman elutriator the centrifugal field employed. Changes be used to elute fractions of cells based
constant that this
in the Beckman J2lC anomolous elution
centrifuge, is related
to
20,
depends in either on size.
than do
fractions not
occur.
centrifuge prevent
collected We
under conclude
conditions that
where the
AND METHODS
All blood used in these experiments was drawn from the same healthy individual. 0003-26Y7/79/130112-04X02.0010 CopyrIght All right,
I’ IY7Y by Academic Prc\\. Inc 01 rrproducr~on ,n ;my term rc\crvrd
I I2
flow
rate of the medium field or the flow rate these variables are held
of cells is eluted. control system
We have found which gave a
well. The elution resulting from this sire ofthe fraction eluted at a particular fractions have a larger size dispersion control
rotor
Centrifugal elutriation is a relatively new procedure for the separation of cells. With this method, the sedimentation of the cells in a centrifugal field is opposed by a counterflow of liquid and changes in either centrifugal field or flow rate can be used to fractionate a cell population. The separation achieved is based on size with small contributions due to shape and density (1,2). Elutriation has several advantages as a method for cell fractionation. It is gentle and requires no gradients. It is more rapid and a relatively higher cell concentration can be employed than with other velocity sedimentation methods. A number of different kinds of cell populations (3-7) have been fractionated using this procedure. We have identified and characterized a problem in the Beckman elutriator system employing a J21C centrifuge which results in a decrease in the effectiveness of this system to resolve cells of different size. MATERIALS
upon the the centrifugal Even when
refrigeration-related
temperature
used with the Beckman elutriation maximum resolution of cells.
MORRISON
1979
a periodic pulse the temperature
periodically pulsed temperature drop in the centrifuge change in temperature caused a shift in the modal cell flow rate and centrifugal field. Because of this. the
Elutriation
produces
temperature system temperature
of
the
fluctuations Beckman
fluctuations
J2lC which
Cells were used within 2 days after being drawn. The erythrocytes were obtained by centrifugation of whole blood at 5OOg for 10 min. The cells were then suspended in isotonic saline and washed three times by centrifugation to obtain cells free of serum proteins. The cells were then suspended to a 50% hematocrit in isotonic saline buffered with 0.01 M phosphate, pH 7.4, containing 0.2% bovine serum albumin (Sigma Fraction V), penicillin, 100 U/ml, Fungizone, 0.25 pgiml, and streptomycin, 100 kg/ml (Gibco antibiotic-antimycotic). This medium was used as the elutriation medium. EluttYtrtiotl. The separation medium was pumped through the system using a Cole Parmer Master flex pump with a No. 7013 pumphead. The pump control box was modified by replacing the normal control potentiometer with a IO-turn potentiometer. This modification allowed a finer, more reproducible control of the pump flow rate. Flow rate was measured with a Brooks doubleball flow valve. Approximately 1.7 x IO” cells in 0.4 ml of buffer were injected through an in-line “y” fitting placed just
RESOLUTION
OF
CELLS
BY
CENTRIFUGAL
ELUTRIATION
113
a dedicated 4K MP-12 with 4K RAM computer. The computer was calibrated with S-pm-diameter latex beads and was programmed to plot sample size vs population as a histogram. The computer also calculated the log mean. mode, and median diameter of the sample. For cell sizing, the sample was drawn through the orifice by a pressure differential of 2.2 cm Hg. RESULTS
FIG. I, Comparison temperature with and regulation. by following
Cell the
of the without
elution from absorbance
temperature was monitored probe placed in the eluant performed with centrifuge (B)
the
perature the flow
elutriation regulation. rate was
elution of cells and eluant centrifuge temperature the rotor at 280
by a recording thermal stream. (A) The elutriation temperature regulation. and
performed Arrows changed.
was monitored nm and eluant
without a-e
indicate
centrifuge points
temwhere
before the rotor inlet. The initial flow rate was 7.7 mlimin and the rotor speed was maintained at 2070 rpm. In order to elute the cells, the flow rate was increa:sed in seven increments from 7.7 to 12.1 mlimin. The last fraction was collected by stopping the rotor with the eluant flowing through the chamber at 13.1 mlimin. The elution of the cells was monitored at 280 nm. Fractions were collected manually at each change in the flow rate. The temperature of the eluant was monitored with a thermocouple sensor in the tubing near the point of exit from the centrifuge. Ccl/ corrr~ti,~~. Immediately after Icollection, the number and size of the cells in the fractions were determined with a Particle Data Company Electrozone Celloscope electrostatic particle counter. For these experiments, a 46pm counting orifice was used and the counter output was fed into
AND DISCUSSION
At a particular setting of the centrifugal field, the cells can be eluted by changing the flow rate of the fluid pumped through the elutriator system. The elution of cells was monitored at 280 nm using a column monitor with a recorder. During the separation of erythrocytes, a periodic pulse of eluted cells was observed, as shown in Fig. 1A. when the temperature was set at 22°C on the J2lC centrifuge used with the Beckman elutriation system. This periodic elution of cells occurred even though there was no change in either the flow rate or the centrifugal field. By carefully monitoring all possible sources which could produce this perturbation, it was found that each time the refrigeration came on, there was an increase in the quantity of cells that eluted. With the temperature set at 22°C. when the refrigeration delivered its cooling pulse, the temperature dropped 4-S’C in the chamber. The temperature drop and slow rise appeared to correlate with an elution of cells. A recording temperature probe placed in the eluant line showed that the elutriation medium temperature was 4°C higher than the chamber temperature and that the medium temperature fluctuations were comparable in timing to cell elution (see Fig. 1A). In contrast, when the centrifuge refrigeration system was turned off and the centrifuge chamber was opened to allow air flow to maintain the chamber temperature at 22°C. the pulsed cell elution was eliminated (see Fig. IB). The recording thermal probe
114
GRANT
AND
in the eluant line showed that under these conditions, although the eluant temperature was elevated 4°C above ambient, the thermal fluctuations had been eliminated. The influence of this temperature fluctuation on the number of cells in each fraction and their size was investigated. The number of cells in each fraction was not greatly distorted by the temperature control artifact. Figure 2A shows that when cell count is plotted vs fraction number, only a small difference in elution profiles is observed. The distribution of cells as a function of flow rate appeared almost normal. However, a very significant difference was found in the size distribution of the cells in the fractions. When the modal volume of the cells in each fraction was plotted vs fraction number (Fig. 2B), a marked difference in modal size of the cells was observed. This meant that with the refrigeration system in use, there was significant decrease in the resolution of cells differing in size. This temperature-related elution of cells was observed with both fixed and unfixed erythrocytes from humans and rats as well as viable nucleated cells. It was also observed in the Sanderson elutriation cell (8). This phenomenon may explain why Sanderson and Bird (2) could not obtain satisfactory fractionation at 4°C. Since this irregularity was observed with both fixed and unfixed erythrocytes, the elution pattern was probably not due to temperature-related changes in cell shape. The temperature-related elution may, however, be explained by changes in the viscosity of the elutriation medium. The log of viscosity (7) is inversely related to the temperature (T in “K): log n = KIT. Thus. at any combination of flow rate and R force in the elutriator rotor. a decrease in temperature which causes a change in the medium viscosity will elute particles of a larger size.
MORRISON
FIG. 2. Comparison of elutriator fractions collected under similar conditions with and without centrifuge temperature regulation. Erythrocyte population in the fractions was measured by electrostatic cell counting. (A) Total cells per fraction, and (B) modal volume of the cells in the fractions. The elutriation was performed with (0) or without t x) centrifuge temperature regulation.
We have experimentally measured the viscosity of the elutriation medium employed in these studies with a falling-ball viscometer. By using the values obtained and assuming that the cells were spherical particles with a mean volume of 90 pm”, we have calculated, using Stokes law, that a change in temperature of 2°C from 27.5 to 2.5.5”C will cause cells of 107 pm” to be eluted. Hence, on a theoretical basis and from our experimental observations, a temperature fluctuation such as we have observed can cause a shift in the modal diameter of the cells eluted at a particular setting and result in a wider dispersion of cell sizes in a given fraction. This temperature regulation problem can be overcome by running the elutriator at room temperature with an open chamber. While this will solve the problem for some
RESOLUTION
OF CELLS
BY CENTRIFUGAL
experiments, in many cases a lower temperature is desirable. Presently. the only solution, which is not very satisfactory, would be to move the centrifuge into a cold room. We are currently investigating other possible solutions to temperature regulation in the Beckman centrifuge so that the optimum resolution of cells may be obtained. ACKNOWLEDGMENTS This work was supported in part by Project Grant AM IX106 from the National Institutes of Health, Cancer Center Support (CORE) Grant CA 21765 from the National Cancer Institute. U. S. Army Contract DAM D-5047. and ALSAC.
ELUTRIATION
115
REFERENCES R. J. (1978) ~!xrc~fi~~is 1, l-8. 2. Sanderson, R. J., and Bird, K. E. (1977) ~Mctlro& Cell Bid. 15, 1- 14. 3. Grabske, R. J.. Lake. S.. Gledhill, B. L.. and Meistrich. M. f 1975) ./. C‘rll Plr).\i<~/. 86, 177190. 4. Knook. D. L.. and Sleyster, E. Ch. I 1976) E.t-1’. I.
Grdbske,
Cell
Rcs.
99,
444-449.
5. Preisler, H. D.. Walczak, J.. Renick. J., and Rustum. Y. M. (19771 Ca/rt.c~r Km. 37, 3876-3880. 6. Meistrich. M. L.. Meyn. R. E.. and Barlogie. B. (1977) E-r/>. (‘e/I Rev. 105, 169- 177. 7. Grdina, D. J.. Peters. L. J.. Jones, S., and Chart. E. (1978) J. Ntrr. Ctrltcvr Inst. 61, 209-Z 14. 8. Sanderson. R. J., Bird. K. E., Palmer, N. F.. and Brenman.J.(1976)A~~cr/.Bir~c~lrc~n~. 71.615-6X.
98.
HIOCHthllSlRY
112-I
Resolution
15 (1979)
of Cells by Centrifugal
WILSON D. GRANT AND MARTIN
Received
Cell and can
February
separation using the Beckman elutriator the centrifugal field employed. Changes be used to elute fractions of cells based
constant that this
in the Beckman J2lC anomolous elution
centrifuge, is related
to
20,
depends in either on size.
than do
fractions not
occur.
centrifuge prevent
collected We
under conclude
conditions that
where the
AND METHODS
All blood used in these experiments was drawn from the same healthy individual. 0003-26Y7/79/130112-04X02.0010 CopyrIght All right,
I’ IY7Y by Academic Prc\\. Inc 01 rrproducr~on ,n ;my term rc\crvrd
I I2
flow
rate of the medium field or the flow rate these variables are held
of cells is eluted. control system
We have found which gave a
well. The elution resulting from this sire ofthe fraction eluted at a particular fractions have a larger size dispersion control
rotor
Centrifugal elutriation is a relatively new procedure for the separation of cells. With this method, the sedimentation of the cells in a centrifugal field is opposed by a counterflow of liquid and changes in either centrifugal field or flow rate can be used to fractionate a cell population. The separation achieved is based on size with small contributions due to shape and density (1,2). Elutriation has several advantages as a method for cell fractionation. It is gentle and requires no gradients. It is more rapid and a relatively higher cell concentration can be employed than with other velocity sedimentation methods. A number of different kinds of cell populations (3-7) have been fractionated using this procedure. We have identified and characterized a problem in the Beckman elutriator system employing a J21C centrifuge which results in a decrease in the effectiveness of this system to resolve cells of different size. MATERIALS
upon the the centrifugal Even when
refrigeration-related
temperature
used with the Beckman elutriation maximum resolution of cells.
MORRISON
1979
a periodic pulse the temperature
periodically pulsed temperature drop in the centrifuge change in temperature caused a shift in the modal cell flow rate and centrifugal field. Because of this. the
Elutriation
produces
temperature system temperature
of
the
fluctuations Beckman
fluctuations
J2lC which
Cells were used within 2 days after being drawn. The erythrocytes were obtained by centrifugation of whole blood at 5OOg for 10 min. The cells were then suspended in isotonic saline and washed three times by centrifugation to obtain cells free of serum proteins. The cells were then suspended to a 50% hematocrit in isotonic saline buffered with 0.01 M phosphate, pH 7.4, containing 0.2% bovine serum albumin (Sigma Fraction V), penicillin, 100 U/ml, Fungizone, 0.25 pgiml, and streptomycin, 100 kg/ml (Gibco antibiotic-antimycotic). This medium was used as the elutriation medium. EluttYtrtiotl. The separation medium was pumped through the system using a Cole Parmer Master flex pump with a No. 7013 pumphead. The pump control box was modified by replacing the normal control potentiometer with a IO-turn potentiometer. This modification allowed a finer, more reproducible control of the pump flow rate. Flow rate was measured with a Brooks doubleball flow valve. Approximately 1.7 x IO” cells in 0.4 ml of buffer were injected through an in-line “y” fitting placed just
RESOLUTION
OF
CELLS
BY
CENTRIFUGAL
ELUTRIATION
113
a dedicated 4K MP-12 with 4K RAM computer. The computer was calibrated with S-pm-diameter latex beads and was programmed to plot sample size vs population as a histogram. The computer also calculated the log mean. mode, and median diameter of the sample. For cell sizing, the sample was drawn through the orifice by a pressure differential of 2.2 cm Hg. RESULTS
FIG. I, Comparison temperature with and regulation. by following
Cell the
of the without
elution from absorbance
temperature was monitored probe placed in the eluant performed with centrifuge (B)
the
perature the flow
elutriation regulation. rate was
elution of cells and eluant centrifuge temperature the rotor at 280
by a recording thermal stream. (A) The elutriation temperature regulation. and
performed Arrows changed.
was monitored nm and eluant
without a-e
indicate
centrifuge points
temwhere
before the rotor inlet. The initial flow rate was 7.7 mlimin and the rotor speed was maintained at 2070 rpm. In order to elute the cells, the flow rate was increa:sed in seven increments from 7.7 to 12.1 mlimin. The last fraction was collected by stopping the rotor with the eluant flowing through the chamber at 13.1 mlimin. The elution of the cells was monitored at 280 nm. Fractions were collected manually at each change in the flow rate. The temperature of the eluant was monitored with a thermocouple sensor in the tubing near the point of exit from the centrifuge. Ccl/ corrr~ti,~~. Immediately after Icollection, the number and size of the cells in the fractions were determined with a Particle Data Company Electrozone Celloscope electrostatic particle counter. For these experiments, a 46pm counting orifice was used and the counter output was fed into
AND DISCUSSION
At a particular setting of the centrifugal field, the cells can be eluted by changing the flow rate of the fluid pumped through the elutriator system. The elution of cells was monitored at 280 nm using a column monitor with a recorder. During the separation of erythrocytes, a periodic pulse of eluted cells was observed, as shown in Fig. 1A. when the temperature was set at 22°C on the J2lC centrifuge used with the Beckman elutriation system. This periodic elution of cells occurred even though there was no change in either the flow rate or the centrifugal field. By carefully monitoring all possible sources which could produce this perturbation, it was found that each time the refrigeration came on, there was an increase in the quantity of cells that eluted. With the temperature set at 22°C. when the refrigeration delivered its cooling pulse, the temperature dropped 4-S’C in the chamber. The temperature drop and slow rise appeared to correlate with an elution of cells. A recording temperature probe placed in the eluant line showed that the elutriation medium temperature was 4°C higher than the chamber temperature and that the medium temperature fluctuations were comparable in timing to cell elution (see Fig. 1A). In contrast, when the centrifuge refrigeration system was turned off and the centrifuge chamber was opened to allow air flow to maintain the chamber temperature at 22°C. the pulsed cell elution was eliminated (see Fig. IB). The recording thermal probe
114
GRANT
AND
in the eluant line showed that under these conditions, although the eluant temperature was elevated 4°C above ambient, the thermal fluctuations had been eliminated. The influence of this temperature fluctuation on the number of cells in each fraction and their size was investigated. The number of cells in each fraction was not greatly distorted by the temperature control artifact. Figure 2A shows that when cell count is plotted vs fraction number, only a small difference in elution profiles is observed. The distribution of cells as a function of flow rate appeared almost normal. However, a very significant difference was found in the size distribution of the cells in the fractions. When the modal volume of the cells in each fraction was plotted vs fraction number (Fig. 2B), a marked difference in modal size of the cells was observed. This meant that with the refrigeration system in use, there was significant decrease in the resolution of cells differing in size. This temperature-related elution of cells was observed with both fixed and unfixed erythrocytes from humans and rats as well as viable nucleated cells. It was also observed in the Sanderson elutriation cell (8). This phenomenon may explain why Sanderson and Bird (2) could not obtain satisfactory fractionation at 4°C. Since this irregularity was observed with both fixed and unfixed erythrocytes, the elution pattern was probably not due to temperature-related changes in cell shape. The temperature-related elution may, however, be explained by changes in the viscosity of the elutriation medium. The log of viscosity (7) is inversely related to the temperature (T in “K): log n = KIT. Thus. at any combination of flow rate and R force in the elutriator rotor. a decrease in temperature which causes a change in the medium viscosity will elute particles of a larger size.
MORRISON
FIG. 2. Comparison of elutriator fractions collected under similar conditions with and without centrifuge temperature regulation. Erythrocyte population in the fractions was measured by electrostatic cell counting. (A) Total cells per fraction, and (B) modal volume of the cells in the fractions. The elutriation was performed with (0) or without t x) centrifuge temperature regulation.
We have experimentally measured the viscosity of the elutriation medium employed in these studies with a falling-ball viscometer. By using the values obtained and assuming that the cells were spherical particles with a mean volume of 90 pm”, we have calculated, using Stokes law, that a change in temperature of 2°C from 27.5 to 2.5.5”C will cause cells of 107 pm” to be eluted. Hence, on a theoretical basis and from our experimental observations, a temperature fluctuation such as we have observed can cause a shift in the modal diameter of the cells eluted at a particular setting and result in a wider dispersion of cell sizes in a given fraction. This temperature regulation problem can be overcome by running the elutriator at room temperature with an open chamber. While this will solve the problem for some
RESOLUTION
OF CELLS
BY CENTRIFUGAL
experiments, in many cases a lower temperature is desirable. Presently. the only solution, which is not very satisfactory, would be to move the centrifuge into a cold room. We are currently investigating other possible solutions to temperature regulation in the Beckman centrifuge so that the optimum resolution of cells may be obtained. ACKNOWLEDGMENTS This work was supported in part by Project Grant AM IX106 from the National Institutes of Health, Cancer Center Support (CORE) Grant CA 21765 from the National Cancer Institute. U. S. Army Contract DAM D-5047. and ALSAC.
ELUTRIATION
115
REFERENCES R. J. (1978) ~!xrc~fi~~is 1, l-8. 2. Sanderson, R. J., and Bird, K. E. (1977) ~Mctlro& Cell Bid. 15, 1- 14. 3. Grabske, R. J.. Lake. S.. Gledhill, B. L.. and Meistrich. M. f 1975) ./. C‘rll Plr).\i<~/. 86, 177190. 4. Knook. D. L.. and Sleyster, E. Ch. I 1976) E.t-1’. I.
Grdbske,
Cell
Rcs.
99,
444-449.
5. Preisler, H. D.. Walczak, J.. Renick. J., and Rustum. Y. M. (19771 Ca/rt.c~r Km. 37, 3876-3880. 6. Meistrich. M. L.. Meyn. R. E.. and Barlogie. B. (1977) E-r/>. (‘e/I Rev. 105, 169- 177. 7. Grdina, D. J.. Peters. L. J.. Jones, S., and Chart. E. (1978) J. Ntrr. Ctrltcvr Inst. 61, 209-Z 14. 8. Sanderson. R. J., Bird. K. E., Palmer, N. F.. and Brenman.J.(1976)A~~cr/.Bir~c~lrc~n~. 71.615-6X.