CI~YOBIOLOGY
12,
98-101
(19%)
An Audio
Frequency Magnetic Tape Programmer for Cryopreservation Equipment
W. J. WEAVER,
F. S. WEBER, M. G. REINEY,
Department of Surgery, Univedty
Copyright
AN righta oY
1975 by Academic Press. Inc. reproduction in any form reserved.
S. W. JACOB
of Oregon Medical School, Portland, Oregon 97201
Long-term storage of cells has become a reality, in part due to the achievement of successful freeze-thaw techniques. Essential to many cryopreservation procedures are versatile and reproducible programs controlling the thermal variables to which the cells are exposed ( l-6). To achieve this, a variety of systems are available for generating voltage/time profiles to act as program analogs; they include mechanical cam followers, electronic and optical profiIe followers, and electronic or electromechanical linear ramp generators with or without following resistance-diode circuits to produce curvilinear relationships ( 7). This paper describes an alternate approach to the problem of generating a sIowly changing dc program for controlled cooling by employing a frequency-to-dc converter programmer, consisting of a standard magnetic cassette tape recorder and a solid state electronic circuit (Fig. 1). This method is useful where complex temperature/time relationships are desired, or where many experimental variations within the programs are used. Components for this system are available commercially and the construction of the circuitry requires no speciaIized tools. An audio tone signal varying in frequency according to the desired temperature/time profile is recorded on a cassette tape with the aid .of an audio generator (Heathkit IG-102, Health Co., Benton Harbor, Mich. ). Received November 29, 1973.
AND
The tape audio output signal is connected to the input of a phase locked loop (PLL) unit (Fig, 1) where it is transformed to a dc voltage output for controIling a cryopreservation cycle. The heart of the PLL unit is an integrated PLL (SE/NE 565, Signetics Corporation, Sunnyvale, Calif. ), A PLL is an electronic servo in which the input frequency is compared with a frequency generated by an integral oscillator, The resulting difference signal is fed back to synchronize the oscillator with the input signal, The feedback loop is then said to be locked, and the feedback signal is thus a dc voltage proportional to the input signal frequency. The device can ,therefore be used as a frequency-to-dc converter within its operating range. Although the discrete (nonintegrated) PLL has been used for years to track satellites, stabilize the frequency of klystrons, and filter information out of noise, application of the PLL to a low cost program system was not practical until the recent introduction of the integrated PLL. The power su~pply for the PLL unit is an UItraminiature 515 VDC module power supply (4.9 x 2.5 x 1.0 cm, BPM-15/X), Date1 Systems, Inc., Canton, Mass. ). All electrical components are packaged in a small (10.5 x 7 x 8 cm) metal utility cabinet (Fig, 1). After the construction of the PLL unit, the circuit (Fig. 2) is calibrated to achieve compatibility with a specific cryogenic cOntroIler requiring a variable input signal of
PROGRAMMER
FOR CRYOPRESERVATION
EQUIPMENT
FIG. 1. Components of a programmer for cryopreservation equipment: loop unit and (B) a standard magnetic cassette tape recorder. either cwrent or voltage. A Leeds and Northrup series 60 controller mated to a Speedmax H, Model S indicating recorder (Leeds and Northrup Co., Sunneytown
99
(A) phase locked
Pike, North Wales, Penna.) is incorporated into this system. With the input termina1 grounded and a digital frequency counter (Healthkit IG-72, Health Co., Benton Har-
FIG. 2. Schematic diagram of the integrated phase locked loop and associated circuit. This circuit functions as a frequency-to-dc converter. Resistors A, B, and C are 10, IO, and 20 K, respectively. Capacitor values are in microfarads unless noted.
WEAVER
FIG. 3. Schematic diagram of a programmed liquid Na cooling apparatus. An audio tone signal varying in frequency according to the desired temperature/time profile is fed into a phase locked loop (PLL) frequency-to-dc converter unit. The control recorder senses the voltage change in the gas/reference cell thermocouples, and compares this with the setpoint reference received from the PLL unit. The difference in the gas/reference cell signal and setpoint reference produces an error signal that regulates the heater control. Liquid ii? flows from the reservoir through a stainless steel tube resistance heater where it is converted to gas of the desired temperature before flowing through the freezing chamber. The heater control output energizes the resistance heater transformer and thus determines the gas temperature. A detailed description of the cooling unit will be reported Iater.
bor, Mich.) connected to the test terminal, the PLL center frequency is set at 1 KHz by adjusting the variable resistor (A). The ground is then removed from the input terminal and potentiometer control (B) is adjusted to meet minimum controller voltage or current input requirements, Adjustment of the variabIe resistor (C) allows the output voltage to be set over a 10-V range, Input signal frequencies above or below the PLL center frequency results in an output of +dc and -dc voltage, respectively. As the input signal frequency reaches the PLL center frequency (1 KHZ), the PLL will achieve “lock” and subsequently will track a signal over its entire “lock” range which is approximately those frequencies between 700 and 1300 Hz.
ET AL.
The tape recorder used (Fig. 1) is: it Panasonic cassette tape recorder (Model RQ-409S, Matsushita Electric Corp. of America, New York, N.Y. ) . Reproducibility of a 1 KHz recorded signal upon replay, using this machine and Sony, ,C60 cassette tape ( Superscope, Sun Valley, Calif. ) showed a frequency variation of tl Hz. This frequency variation appeared to be transient and equally distributed above and below the recorded signal, thus reducing the effect of the variation, Measurement of the PLL unit dc voltage output produced by a program repeatedly generated from the same tape is linear and has a variation of less than 1%. An example of the incorporation of the programmer in a cryopreservation system is schematically diagramed in Fig. 3. This equipment is presently being employed in canine lymphocyte freezing studies. The program will take as long to produce as the process that is to be controlled. However, this inconvenience to the investigator is compensated for by his ability, during a cryogenic procedure, to follow manually a time/temperature curve previously drawn on the chart of a strip-chart recorder. To do this the audio generator is connected directly to the PLL unit as well as the tape recorder. Since the audio generator output is recorded on the cassette tape simultaneously with the manual operation, replaying th e cassette tape reproduces the original program. In this configuration when changes are made in experimental procedures, e.g., in the apparatus, the geometry of the sampIe, or the perfusate composition, the flow rate and pressur+the need to produce a suitable time/temperature program by trial and error, as described by Ellis et al. ( 8)) is eliminated. With the use of additional PLL units, an investigator working with an organ perfusion system could place more than one program on a single tape, and thus control such parameters as perfusion pressure, pH and flow, concurrently, To achieve this would require multiple channel recording
PROGRAMMER
FOR CRYOPRESERVATION
equipment or superimposition of nonharmanic audio signals On a single channel without simultaneous erasing. In the latter instance setting the “lock” range of an individual PLL unit to correspond with its respective audio signals will subsequently permit separation of the programs. SUMMARY
The application of a magnetic cassette recorder and a simple solid state circuit as a programmer for cryopreservation equipment is described. Advantages of this system in&de: (I ) ease and Rexibihty in programming complex time/temperature relationships; (2) incorporation of many experimental variations into single programs. ACKNOWLEDGMENTS This work was supported by Grant AM09136-14 from the National Institutes of Health. REFERENCES 1. Mazur, P. CryobioIogy: The freezing of biological systems, Science 168, 939-949 (1970).
EQUIPMENT
101
2. Hoki, A. Types of freezing arid the post-thawing survival of mammalian tissue. K&e J. Merl. Sci. 13, 67-79 ( 1967). 3. Takehara, I., and Rowe, A. W. Increase in ATPase activity in red cell membranes as a function of freezing regimen. CTyobioZogy 8, 559-565 (1971). 4. Carter, J. E., Riscb, S. J., Graham, E. F., and Lillehei, R. C. The effect of pressure and cooling rate on spermatozoa, kidney tissue, and the whole kidney. Cryobiulogy IO, 263270 ( 1973). 5. Albrecht, R. M., Omdorff, G. R., and MacKenzie, A. P. Survival of certain microorganisms subjected to rapid and very rapid freezing on membrane filters. Cry&o&y 10, 233-239 (1973). 6. Robertson, R. D., and Jacob, S. W. The preservation of intact organs. Ado. Surg. 3, 75-159 (1968). 7. Pegg, D. E., Hayes, A. R., and Kingston, R.E. Cooling equipment for use in cryopreservation. Cryobiology 10, 271-281 ( 1973). 8. EIlis, M. J., Davies, P. W., and Hobbs, K. E. F. A programmed cooling unit far experimental organ preservation. Cryobiology 11, 100-103 (1974).
12,
98-101
(19%)
An Audio
Frequency Magnetic Tape Programmer for Cryopreservation Equipment
W. J. WEAVER,
F. S. WEBER, M. G. REINEY,
Department of Surgery, Univedty
Copyright
AN righta oY
1975 by Academic Press. Inc. reproduction in any form reserved.
S. W. JACOB
of Oregon Medical School, Portland, Oregon 97201
Long-term storage of cells has become a reality, in part due to the achievement of successful freeze-thaw techniques. Essential to many cryopreservation procedures are versatile and reproducible programs controlling the thermal variables to which the cells are exposed ( l-6). To achieve this, a variety of systems are available for generating voltage/time profiles to act as program analogs; they include mechanical cam followers, electronic and optical profiIe followers, and electronic or electromechanical linear ramp generators with or without following resistance-diode circuits to produce curvilinear relationships ( 7). This paper describes an alternate approach to the problem of generating a sIowly changing dc program for controlled cooling by employing a frequency-to-dc converter programmer, consisting of a standard magnetic cassette tape recorder and a solid state electronic circuit (Fig. 1). This method is useful where complex temperature/time relationships are desired, or where many experimental variations within the programs are used. Components for this system are available commercially and the construction of the circuitry requires no speciaIized tools. An audio tone signal varying in frequency according to the desired temperature/time profile is recorded on a cassette tape with the aid .of an audio generator (Heathkit IG-102, Health Co., Benton Harbor, Mich. ). Received November 29, 1973.
AND
The tape audio output signal is connected to the input of a phase locked loop (PLL) unit (Fig, 1) where it is transformed to a dc voltage output for controIling a cryopreservation cycle. The heart of the PLL unit is an integrated PLL (SE/NE 565, Signetics Corporation, Sunnyvale, Calif. ), A PLL is an electronic servo in which the input frequency is compared with a frequency generated by an integral oscillator, The resulting difference signal is fed back to synchronize the oscillator with the input signal, The feedback loop is then said to be locked, and the feedback signal is thus a dc voltage proportional to the input signal frequency. The device can ,therefore be used as a frequency-to-dc converter within its operating range. Although the discrete (nonintegrated) PLL has been used for years to track satellites, stabilize the frequency of klystrons, and filter information out of noise, application of the PLL to a low cost program system was not practical until the recent introduction of the integrated PLL. The power su~pply for the PLL unit is an UItraminiature 515 VDC module power supply (4.9 x 2.5 x 1.0 cm, BPM-15/X), Date1 Systems, Inc., Canton, Mass. ). All electrical components are packaged in a small (10.5 x 7 x 8 cm) metal utility cabinet (Fig, 1). After the construction of the PLL unit, the circuit (Fig. 2) is calibrated to achieve compatibility with a specific cryogenic cOntroIler requiring a variable input signal of
PROGRAMMER
FOR CRYOPRESERVATION
EQUIPMENT
FIG. 1. Components of a programmer for cryopreservation equipment: loop unit and (B) a standard magnetic cassette tape recorder. either cwrent or voltage. A Leeds and Northrup series 60 controller mated to a Speedmax H, Model S indicating recorder (Leeds and Northrup Co., Sunneytown
99
(A) phase locked
Pike, North Wales, Penna.) is incorporated into this system. With the input termina1 grounded and a digital frequency counter (Healthkit IG-72, Health Co., Benton Har-
FIG. 2. Schematic diagram of the integrated phase locked loop and associated circuit. This circuit functions as a frequency-to-dc converter. Resistors A, B, and C are 10, IO, and 20 K, respectively. Capacitor values are in microfarads unless noted.
WEAVER
FIG. 3. Schematic diagram of a programmed liquid Na cooling apparatus. An audio tone signal varying in frequency according to the desired temperature/time profile is fed into a phase locked loop (PLL) frequency-to-dc converter unit. The control recorder senses the voltage change in the gas/reference cell thermocouples, and compares this with the setpoint reference received from the PLL unit. The difference in the gas/reference cell signal and setpoint reference produces an error signal that regulates the heater control. Liquid ii? flows from the reservoir through a stainless steel tube resistance heater where it is converted to gas of the desired temperature before flowing through the freezing chamber. The heater control output energizes the resistance heater transformer and thus determines the gas temperature. A detailed description of the cooling unit will be reported Iater.
bor, Mich.) connected to the test terminal, the PLL center frequency is set at 1 KHz by adjusting the variable resistor (A). The ground is then removed from the input terminal and potentiometer control (B) is adjusted to meet minimum controller voltage or current input requirements, Adjustment of the variabIe resistor (C) allows the output voltage to be set over a 10-V range, Input signal frequencies above or below the PLL center frequency results in an output of +dc and -dc voltage, respectively. As the input signal frequency reaches the PLL center frequency (1 KHZ), the PLL will achieve “lock” and subsequently will track a signal over its entire “lock” range which is approximately those frequencies between 700 and 1300 Hz.
ET AL.
The tape recorder used (Fig. 1) is: it Panasonic cassette tape recorder (Model RQ-409S, Matsushita Electric Corp. of America, New York, N.Y. ) . Reproducibility of a 1 KHz recorded signal upon replay, using this machine and Sony, ,C60 cassette tape ( Superscope, Sun Valley, Calif. ) showed a frequency variation of tl Hz. This frequency variation appeared to be transient and equally distributed above and below the recorded signal, thus reducing the effect of the variation, Measurement of the PLL unit dc voltage output produced by a program repeatedly generated from the same tape is linear and has a variation of less than 1%. An example of the incorporation of the programmer in a cryopreservation system is schematically diagramed in Fig. 3. This equipment is presently being employed in canine lymphocyte freezing studies. The program will take as long to produce as the process that is to be controlled. However, this inconvenience to the investigator is compensated for by his ability, during a cryogenic procedure, to follow manually a time/temperature curve previously drawn on the chart of a strip-chart recorder. To do this the audio generator is connected directly to the PLL unit as well as the tape recorder. Since the audio generator output is recorded on the cassette tape simultaneously with the manual operation, replaying th e cassette tape reproduces the original program. In this configuration when changes are made in experimental procedures, e.g., in the apparatus, the geometry of the sampIe, or the perfusate composition, the flow rate and pressur+the need to produce a suitable time/temperature program by trial and error, as described by Ellis et al. ( 8)) is eliminated. With the use of additional PLL units, an investigator working with an organ perfusion system could place more than one program on a single tape, and thus control such parameters as perfusion pressure, pH and flow, concurrently, To achieve this would require multiple channel recording
PROGRAMMER
FOR CRYOPRESERVATION
equipment or superimposition of nonharmanic audio signals On a single channel without simultaneous erasing. In the latter instance setting the “lock” range of an individual PLL unit to correspond with its respective audio signals will subsequently permit separation of the programs. SUMMARY
The application of a magnetic cassette recorder and a simple solid state circuit as a programmer for cryopreservation equipment is described. Advantages of this system in&de: (I ) ease and Rexibihty in programming complex time/temperature relationships; (2) incorporation of many experimental variations into single programs. ACKNOWLEDGMENTS This work was supported by Grant AM09136-14 from the National Institutes of Health. REFERENCES 1. Mazur, P. CryobioIogy: The freezing of biological systems, Science 168, 939-949 (1970).
EQUIPMENT
101
2. Hoki, A. Types of freezing arid the post-thawing survival of mammalian tissue. K&e J. Merl. Sci. 13, 67-79 ( 1967). 3. Takehara, I., and Rowe, A. W. Increase in ATPase activity in red cell membranes as a function of freezing regimen. CTyobioZogy 8, 559-565 (1971). 4. Carter, J. E., Riscb, S. J., Graham, E. F., and Lillehei, R. C. The effect of pressure and cooling rate on spermatozoa, kidney tissue, and the whole kidney. Cryobiulogy IO, 263270 ( 1973). 5. Albrecht, R. M., Omdorff, G. R., and MacKenzie, A. P. Survival of certain microorganisms subjected to rapid and very rapid freezing on membrane filters. Cry&o&y 10, 233-239 (1973). 6. Robertson, R. D., and Jacob, S. W. The preservation of intact organs. Ado. Surg. 3, 75-159 (1968). 7. Pegg, D. E., Hayes, A. R., and Kingston, R.E. Cooling equipment for use in cryopreservation. Cryobiology 10, 271-281 ( 1973). 8. EIlis, M. J., Davies, P. W., and Hobbs, K. E. F. A programmed cooling unit far experimental organ preservation. Cryobiology 11, 100-103 (1974).
