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Fundamental Cryobiology of Human Hematopoietic Progenitor Cells I: Osmotic Characteristics and Volume Distribution
36, 40 – 48 (1998) CY972060
CRYOBIOLOGY ARTICLE NO.
Fundamental Cryobiology of Human Hematopoietic Progenitor Cells I: Osmotic Characteristics and Volume Distribution1 D. Y. Gao,*,† Q. Chang,‡ C. Liu,* K. Farris,* K. Harvey,‡ L. E. McGann,*,§ D. English,‡ J. Jansen,‡ and J. K. Critser*,¶,\,2 *Cryobiology Research Institute and ‡Bone Marrow Transplantation Program, Methodist Hospital of Indiana, Inc., Indianapolis, Indiana 46202 U.S.A.; †Department of Mechanical Engineering, Purdue University, Indianapolis, Indiana 46202 U.S.A.; §Department of Pathology, University of Alberta, Edmonton, Alberta, Canada; ¶ Department of Physiology & Biophysics and Obstetrics & Gynecology and Pediatrics, Indiana University School of Medicine, Indianapolis, Indiana 46202 U.S.A.; and \Department of Veterinary Clinical Sciences and Hillenbrand Biomedical Engineering Center, Purdue University, West Lafayette, Indiana 47907 U.S.A. While methods for the cryopreservation of hematopoietic stem cells are well established, new sources of progenitor cells, such as umbilical cord blood, fetal tissue, and ex vivo expanded progenitor cells, may require refined protocols to achieve optimal recovery after freezing. To predict optimal protocols for cryopreservation of human hematopoietic progenitors, knowledge of fundamental cryobiological characteristics including cell osmotic characteristics, water and cryoprotectant permeability coefficients of cell membrane, and activation energies of these coefficients is required. In this study, we used CD341CD332 cells isolated from human bone marrow as hematopoietic progenitor cell models/representatives to study the osmotic characteristics of the progenitor cells. Volume distribution and osmotic behavior of the CD341CD332 cells were determined using two different methods: (a) a shape-independent electronic sizing technique and (b) a shape-dependent optical image analysis. The cell diameter was measured to be 8.2 6 1.1 mm (mean 6 SD, n 5 1,091,475, the number of donors 5 8) using the electronic sizing technique or 8.7 6 1.2 mm (mean 6 SD, n 5 1508, the number of donors 5 6) by image analysis at initial (isotonic) osmolality, 325 mosm/kg. The cell volume change was measured after the cells were exposed and equilibrated to different anisosmotic conditions. The cell volume was found to be a linear function of the reciprocal of the extracellular osmolality (Boyle van’t Hoff plot) ranging from 163 to 1505 mosm/kg. The volume fraction of intracellular water which is osmotically active was determined to be 79.5% of the cell volume. It was concluded that human CD341CD332 cells osmotically behave as ideal osmometers. This information coupled with cell water and cryoprotectant permeability coefficients as well as their activation energies (to be determined in the ongoing research projects) will be used to design optimum conditions for cryopreservation of human hematopoietic progenitor cells. © 1998 Academic Press Key Words: human hematopoietic progenitors; CD341CD332 cells; osmotic behavior; Boyle van’t Hoff plot.
Autologous bone marrow transplantation permits the use of myeloablative chemotherapeutic regimens in the treatment of cancer. The success of the therapy depends on viable hematopoietic stem cells after cryopreservation. Although successful cryopreservation of bone marrow was reported over 30 years ago (14) and
provides the basis for autologous bone marrow transplantation (4, 12, 17, 36, 40), protocols for cryopreservation have been developed empirically without an understanding of the fundamental cryobiology of human hematopoietic progenitor/stem cells. (It should be noted that Leibo et al. (13) did a careful and excellent investigation on cooling rate dependence of the survival of mouse bone marrow stem cells.) Therefore, questions pertaining to the fundamental cryobiological characteristics of hematopoietic progenitor cells have not been answered. Because the empiric development of cryopreservation techniques has acceptable results when a large volume of bone marrow cells
Received August 1, 1996; accepted October 1, 1997. 1 This work was supported by Methodist Hospital of Indiana, Inc. 2 To whom correspondence should be addressed at Cryobiology Research Institute, Room 204, Potter Building, Hillenbrand Biomedical Engineering Center, Purdue University, West Lafayette, IN 47907. Fax: (765) 494-1193. Email: [email protected]. 40 0011-2240/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.
OSMOTIC BEHAVIOR OF HEMATOPOIETIC PROGENITORS
are processed, the clinical consequences of cryobiology research have been perceived as minor. With new techniques making it possible to isolate small fractions of pluripotent cells, new cryopreservation technology likely will be required, as the proportional injury in the small cell population may result in nonviable transplant preparations. In addition, cryopreservation of purified hematopoietic stem cells is likely to become a key technology in the upcoming era of gene transfer therapy, and optimizing cell cryosurvival rate is critically important. New sources of progenitor cells, such as umbilical cord blood (3), fetal liver (34), and cells generated by ex vivo expansion (44), may require refined protocols to obtain optimal cryosurvival based on the potential difference in their cryobiological characteristics. The major steps used in cryopreservation of cells can be summarized as follows: (i) adding cryoprotective agents (CPAs) to cells before cooling; (ii) spontaneously or artificially inducing ice crystals in the cell suspension at or a few degrees below the freezing point of the cell suspension, and cooling the cells toward a low temperature at which the cells are stored; (iii) warming the cells; and (iv) removing the CPAs from the cells after thawing the sample. Injury to cryopreserved cells has been shown to be caused by each or a combination of the above steps. For example, cells may be damaged by either severe cell volume excursion (osmotic injury) or potential toxicity of the CPAs (8, 21, 37) in steps i and iv. Intracellular ice formation (5–7, 11, 16, 18 –20, 22–25, 29 –31, 35, 39, 43) during the cooling process (in step ii) can kill cells, and the subsequent recrystallization of the intracellular ice during the warming process (in step iii) can also cause or enhance cell injury (25). Based on the previous investigations (8, 21, 23, 25, 30, 32, 42), one needs to know at least the following information to design optimal cryopreservation protocols: (a) cell osmotic characteristics; (b) permeability of the cell to water (Lp) and to the CPAs (PCPA); (c) activation energies (Ea) of Lp and PCPA, respectively;
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(d) ratio of the cell surface area to volume; (e) intracellular ice formation phenomena; and (f) osmol of solutes initially in the cell. Parameters a– e are cell-type-dependent properties while parameter f is dependent on the concentration and nature of the CPAs. Parameters a– e can be determined from experimental measurements. However, to date, these parameters remain unknown for human hematopoietic progenitor cells. The CD34 antigen, a known progenitor cell marker, is expressed by 1 to 4% of human bone marrow mononuclear cells, while the CD33 antigen, a marker associated with myeloid commitment, is expressed on a higher portion of bone marrow mononuclear cells including a sizable percentage of CD341 cells. CD341CD332 cells include virtually all hematopoietic long-term repopulating cells detected by in vitro assays (1). In the present study, the CD341CD332 cell population isolated from human bone marrow was used as a human hematopoietic progenitor cell model/representative to determine the osmotic characteristics, volume distribution, and osmotically active water volume of the progenitor cells. MATERIALS AND METHODS
Isolation of Human Hematopoietic CD341CD332 Cells Human bone marrow was obtained from 12 healthy donors. Isolated hematopoietic progenitor cells, CD341CD332 cells, were obtained after processing the bone marrow by density gradient centrifugation, soybean agglutinin (SBA), CD341 cell enrichment, and fluorescence-activated cell sorting (1, 3, 4, 17, 40, 41). Leukocyte-rich plasma was obtained after enhanced sedimentation of heparinized bone marrow. This leukocyte-rich product was carefully layered on cushions of Ficoll–Hypaque (density 5 1.077 g/ml) (Sigma, St. Louis, MO). The gradient tubes were then centrifuged at 850g for 30 min and the mononuclear leukocyte layer was aspirated. Mononuclear leukocyte suspensions collected after Ficoll–Hypaque
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were centrifuged and resuspended in Dulbecco’s phosphate-buffered saline without Ca21 or Mg21 (DPBS2) containing 0.5% human gamma globulin (Cohn Fraction II; Sigma) at a concentration of 5 3 106 cells/ml. The cell suspension was placed into AIS MicroCollector T-25 flasks coated with soybean agglutinin (Applied Immune Sciences, Inc., Santa Clara, CA). Each flask contained a total of 2 3 107 mononuclear leukocytes. After a 1-h incubation period at room temperature to ensure sufficient adherence to the flasks, the flasks were gently rocked to dislodge nonadherent cells. These SBA2 cells were aspirated, pooled, centrifuged, and resuspended in DPBS2 containing 0.5% human gamma globulin at a concentration of 5 3 106 cells/ml. For CD341 panning, the cells were collected as described above and placed in the AIS T-25 flasks coated with Murine monoclonal anti-CD341 antibody (clone ICH-3). Following a 1-h incubation, nonadherent cells were removed while the adherent cells were physically dislodged from the AIS T-25 flasks. Cells selected by the AIS technique were typically 20 – 40% positive for the CD341 antigen. A significant and variable portion of the CD341 cells also possessed the CD33 differentiation antigen. These cells were pooled, concentrated by centrifugation, and labeled with two different antibodies for fluorescent analysis and subsequent cell sorting. For cell sorting, cells were labeled with monoclonal anti-CD34 (clone 8G12) (Becton Dickinson, San Jose, CA), directly conjugated with phycoerythrin and anti-CD33 conjugated with fluoroisothiocyanate (Dako, Carpenteria, CA). In these experiments, two different clones of monoclonal anti-CD34 were used in order to optimize the signal of the CD34 antigen by recognizing two different epitopes. The antibodies were simultaneously incubated with the cells for 20 min at 4°C in the dark. Following labeling, the cells were washed once in phosphate-buffered saline (Sigma) containing 1% fetal calf serum (Hyclone, Logan, UT) and 0.1% sodium azide (Sigma). Cells were resuspended in a volume of 3– 4 ml in the wash buffer. The suspension
was placed in the dark at 4°C until further use. Fluorescence-activated cell sorting was accomplished using a FACStar PLUS flow cytometer (Becton Dickson) equipped with a water-cooled argon laser emitting at 488 nm. Subsequent analysis of the cells was performed using Lysis II software (Becton Dickinson). Sorted cells were collected in 12 3 75 mm borosilicate glass tubes (Fisher Scientific, Pittsburg, PA). Typically, the purity of sorted CD341CD332 cells exceeded 97% as determined by the flow cytometer. The purified cells were maintained in M199 medium (Gibco Laboratories, Grand Island, NY) supplemented with 20% (v/v) fetal calf serum at 4°C before treatments and measurements. Preparation of Anisosmotic Cell Suspensions The osmolality of the M199 medium supplemented with 20% (v/v) fetal calf serum was measured to be 325 mosm/kg using an osmometer (Model 3D2; Advanced DigiMatic Osmometer, Needham Heights, MA). This osmolality was used as the initial osmolality of the cells and assumed to be isosmolality. Hyperosmotic solutions were produced by the addition of sucrose to the supplemented M199 medium, and hyposomotic solutions were made by the dilution of the supplemented M199 medium with reagent grade water (Millipore, Burlington, MA). The CD341CD332 cells were exposed to the anisosmotic solutions and allowed to equilibrate with anisosmotic environments for 5 min at room temperature before volume measurement. The osmolality in each final cell suspension was measured. The final osmolalities of anisosmotic cell suspensions were measured to be 163, 243, 270, 325, 585, 925, 1280, and 1505 mosm/kg, respectively. Determination of CD341CD332 Cell Volume Optical image analysis method. Each of the CD341CD332 cell suspensions, with a known osmolality, was placed into 20-mm-deep MicroCell chambers (Conception Technologies, La Jolla, CA). The cells were videotaped using an Olympus BH-2 microscope (Scientific Supply, Schiller Park, IL) with a 20X objective and a
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OSMOTIC BEHAVIOR OF HEMATOPOIETIC PROGENITORS
video camera (CRYOResources, Ltd., New York, NY) connected to both a Panasonic AG6300 VCR and a video processor image analysis system (Motion Analysis VP320, Santa Rosa, CA). The processing assigned pixels at a manually determined light threshold for an accurate outline of the cell. The software, Expert Vision 2.24X (Motion Analysis), was run on an Amdek System 386 PC for use by the Dynamic Morphology System Software package (DMS, Motion Analysis). Assuming a spherical shape for the CD341CD332 cells, the software was used to generate five still frames of each cell for which an average radial length (the mean length of the radius from the centroid to the boundary over all the pixels used as an outline) was calculated. The volume of standard spherical beads with known diameters (Standard Beads, Coulter Electronics, Ltd., Luton, UK) was measured and used as a control to calibrate the measured geometric dimensions of the CD341CD332 cells. Electronic counter method. The CD341CD332 cell suspensions were placed in the sample chamber of an electronic sizing instrument (Coulter Counter, Model ZM; Coulter Electronics, Ltd.). Using a software package (Average Software, Great Canadian Computer Co., Edmonton, Alberta, Canada), the electronic signal (which is a function of cell volume) of each individual cell that moves through the orifice of the Coulter counter was digitized and analyzed by a microcomputer to determine the cell volume (9, 15, 28). The volumes of over 12,000 CD341CD332 cells per donor were measured using the Coulter Counter. The volume distribution of the cells was determined by using Volume-Software (Great Canadian Computer Co.). The electronic signals of standard spherical beads with known diameters (Coulter Electronics, Ltd.) were measured in a standard isotonic solution, Isoton2 (Fisher Scientific), as a control to calibrate the measured volumes of the CD341CD332 cells. Because the number of CD341CD332 cells from each donor was limited (,500,000 cells per donor sample) and a large number of cells is required for the electronic counter measurement, the electronic
TABLE 1 Geometric Dimensions of Human Hematopoietic CD341CD332 Cells at 325 mosm/kg Diameter Volume Surface (mean 6 SD) (mean) area (mean) Number of mm mm3 mm2 cells Method 8.2 6 1.1 8.7 6 1.2 a b
289 345
211 238
1,091,475a Coulter 1,508b Optical
Obtained from 8 different donors. Obtained from 6 different donors.
counter measurement was conducted only at 925, 325, and 243 mosm/kg, respectively. Ideal Osmometer Hypothesis It was hypothesized that the CD341CD332 cells behaved as ideal osmometers, which means the cell volume changes linearly as a function of the reciprocal of the osmolality of the external solution, i.e., the Boyle van’t Hoff (BVH) relationship (2). The mathematical expression of the BVH relationship is V/V i 5 ~1 2 V b /V i !M i /M 1 V b /V i , where V is the cell volume at osmolality M, Vi is the cell volume at isosmolality Mi (325 mOsm in this study), and Vb is the osmotically inactive cell volume (including osmotically inactive intracellular water and nonaqueous/solid part of the cells). Statistical Analysis Experimental data were analyzed and expressed as means 6 SD. Linear regression analysis was used to analyze the Boyle van’t Hoff plot. A Student’s t test was used to examine the difference between the human hematopoietic progenitor cell mean volumes measured by the two different methods (i.e., the optical and Coulter Counter methods) assuming that the data population follows a normal distribution (27). RESULTS
Geometric dimensions (diameter, volume, and membrane surface area) of the human
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CD341CD332 cells at 325 mosm/kg were determined using the optical image analysis and Coulter Counter methods (Table 1). In the optical method, cell diameter was measured, and the cell volume and surface area were calculated assuming the sphericity of cells. In the Coulter technique, cell volume was measured and cell diameter and surface area were calculated, again assuming the sphericity of cells. The optical image analysis showed that the CD341CD332 cells in the osmotic range 136 to 1505 mosm/kg maintained a spherical shape. The mean diameter of all cells measured by optical image analysis (8.7 6 1.2 mm) was consistent with that determined by the Coulter Counter technique (8.2 6 1.1 mm). There was no statistical difference between the two mean values (P . 0.5). In addition, the mean value of the cell diameter was donor-independent (P . 0.5). Although the resolution of the equipment used and the assumption of the spherical shape of the cells (in the optical imaging method) might create some errors in measurement, the results obtained using the different methods showed a similar volume distribution pattern for the CD341CD332 cell population. Figure 1 shows cell volume distributions of six samples, obtained from six different donors, determined by the Coulter Counter technique. Figure 2 shows a similar pattern of cell volume distribution determined by optical image analysis (the number of donors 5 6). In the Coulter Counter measurement, it was assumed that any particle with a diameter less than 6 mm (113 mm3 in volume) was debris and any particle with a diameter greater than 10 mm (523 mm3 in volume) was a cell aggregate (i.e., more than two cells moving simultaneously through the orifice in the Coulter Counter device). These particles, which consist of 0.1% of the total cells selected, were excluded in determining the dimensions of the CD341CD332 cells. The CD341CD332 cell volumes (relative to isotonic cell volume) versus the reciprocal of the external osmolality (i.e., Boyle van’t Hoff plot) are shown in Fig. 3. A linear fit (V/Vi 5 0.886 (325/M) 1 0.205; r2 5 0.98) was obtained using a least-square fit method (Sig-
maPlot, Jandel Scientific Corp., San Rafael, CA). The linearity of the Boyle van’t Hoff plot indicates that the CD341CD332 cells act as ideal osmometers in the range of 163 to 1505 mosm/kg. The nonzero (V/Vi) axis intercept of 0.205 indicates that 20.5% of the cell volume is osmotically inactive. DISCUSSION
The optical image analysis is one of the most commonly used approaches in measuring cell dimensions. This technique can be used to measure the static cell volume or the kinetic volume change of given individual cells. Only a few cells are required for this technique. However, cell shape dependence and time consumption in the image analysis as well as limitation of the optical resolution are disadvantages of the optical image method. The electronic counter technique used in this study is a cell-shapeindependent approach and has been shown to be a very reliable tool to measure the static volume distribution and kinetic volume change of a large number of cells regardless of spheric shape (9, 15, 28). Results generated from both techniques agree well with each other. From this study, it is known that the mean volume of the human hemetopoietic progenitor cells is approximately 289 (by the optical method) or 345 mm3 (by the Coulter Counter method), as indicated in Table 1. These values are comparable with the human lymphocyte mean volume, 290 mm3, as determined by using Coulter Counter (10). The present study also indicates that 20.5% of the human hematopoietic cell volume is osmotically inactive (or, in other words, 79.5% of the cell volume is osmotically active water), which is lower than that (32– 36%) of the human lymphocytes (10, 13). Based on the Boyle van’t Hoff plot, it is concluded that human CD341CD332 cells osmotically behave as ideal osmometers. The information obtained from the present study concerning the CD341CD332 cell volume distribution, cell osmotically inactive volume, and osmotic characteristics provides a basis for ongoing investigations to determine the hematopoietic progenitor cell water and cryoprotectant permeability co-
OSMOTIC BEHAVIOR OF HEMATOPOIETIC PROGENITORS
FIG. 1. Coulter Counter-determined volume distributions of six human hematopoietic CD341CD332 cell samples obtained from six different donors (n 5 6).
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FIG. 2. Volume distribution of human hematopoietic CD341CD332 cells determined by optical image analysis (number of cells measured 5 1508 cells from six donors).
FIG. 3. The CD341CD332 cell volume (relative to isotonic cell volume) versus the reciprocal of the extracellular osmolality (Boyle van’t Hoff plot). V expresses the total cell volume at osmolality M; Vi is the cell volume at isosmolality Mi (325 mosm/kg). Two data points indicated by arrows were determined by the Coulter Counter method. The other data points were determined by using optical image analysis. A linear fit (V/Vi 5 0.87(325/M) 1 0.21; r2 5 0.97) was obtained using a least-square fit method.
OSMOTIC BEHAVIOR OF HEMATOPOIETIC PROGENITORS
efficients and their activation energies. These cell biophysical parameters are required and essential to lead to a better understanding of the fundamental cryobiology of the progenitor cells and to permit the development of optimum procedures for cryopreservation.
10.
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35. Rapatz, G., Nath, J., and Luyet, B. Electron microscope study of erythrocytes in rapidly frozen mammalian blood. Biodynamica 9, 83–96 (1963). 36. Rowley, S. D. Techniques of bone marrow and stem cell cryopreservation and storage. In ‘‘Marrow Transplantation: Practice and Technical Aspects of Stem Cell Reconstitution’’ (R. A. Sacher and J. P. AuBuchon, Eds.), pp. 105–118. Am. Assoc. of Blood Banks, Bethesda, MD, 1992. 37. Schneider, U., and Mazur, P. Osmotic consequences of cryoprotectant permeability and its relation to the survival of frozen-thawed embryos. Theriogenology 21, 68 – 81 (1988). 38. Sputtek, A., and Korber, C. Cryopreservation of red blood cells, platelets, lymphocytes, and stem cells. In ‘‘Clinical Applications of Cryobiology’’ (B. J. Fuller and B. W. W. Grout, Eds.), pp. 96 –112. CRC Press, Boca Raton, FL, 1991. 39. Steponkus, P. L., and Wiest, S. C. Freeze-thaw induced lesions in the plasma membrane. In ‘‘Low Temperature Stress in Crop Plants: The Role of the Membrane’’ (J. M. Lyons, D. G. Graham, and J. K. Raison, Eds.), pp. 231–258. Academic Press, New York, 1979. 40. Stiff, P. J., Koester, A. R., Weidner, M. K., Dvorak, K., and Risher, R. I. Autologous bone marrow transplantation using unfractionated cells cryopreserved in dimethylsulfoxide and hydroxyethyl starch without controlled-rate freezing. Blood 70, 974 –983 (1987). 41. Stiff, P. J., Murgo, A. J., and Zaroulis, C. G. Unfractionated marrow cell cryopreservation of myeloid stem cells from human bone marrow. Cryobiology 20, 17–39 (1983). 42. Toner, M., Cravalho, E., and Karel, M. Cellular response of mouse oocytes to freezing stress: Prediction of intracellular ice formation. J. Biomech. Eng. 115, 169 –181 (1990). 43. Trump, B. F., Young, D. F., Arnold, E. A., and Stowell, R. E. Effects of freezing and thawing on the structure, chemical constitution and function of cytoplasmic structures. FASEB Fed. Proc. 24(Suppl. 15), S144 (1965). 44. Williams, D. A. Ex vivo expansion of hematopoietic stem and progenitor cells—Robbing Peter to pay Paul? Blood 81, 3169 –3172 (1993).
CRYOBIOLOGY ARTICLE NO.
Fundamental Cryobiology of Human Hematopoietic Progenitor Cells I: Osmotic Characteristics and Volume Distribution1 D. Y. Gao,*,† Q. Chang,‡ C. Liu,* K. Farris,* K. Harvey,‡ L. E. McGann,*,§ D. English,‡ J. Jansen,‡ and J. K. Critser*,¶,\,2 *Cryobiology Research Institute and ‡Bone Marrow Transplantation Program, Methodist Hospital of Indiana, Inc., Indianapolis, Indiana 46202 U.S.A.; †Department of Mechanical Engineering, Purdue University, Indianapolis, Indiana 46202 U.S.A.; §Department of Pathology, University of Alberta, Edmonton, Alberta, Canada; ¶ Department of Physiology & Biophysics and Obstetrics & Gynecology and Pediatrics, Indiana University School of Medicine, Indianapolis, Indiana 46202 U.S.A.; and \Department of Veterinary Clinical Sciences and Hillenbrand Biomedical Engineering Center, Purdue University, West Lafayette, Indiana 47907 U.S.A. While methods for the cryopreservation of hematopoietic stem cells are well established, new sources of progenitor cells, such as umbilical cord blood, fetal tissue, and ex vivo expanded progenitor cells, may require refined protocols to achieve optimal recovery after freezing. To predict optimal protocols for cryopreservation of human hematopoietic progenitors, knowledge of fundamental cryobiological characteristics including cell osmotic characteristics, water and cryoprotectant permeability coefficients of cell membrane, and activation energies of these coefficients is required. In this study, we used CD341CD332 cells isolated from human bone marrow as hematopoietic progenitor cell models/representatives to study the osmotic characteristics of the progenitor cells. Volume distribution and osmotic behavior of the CD341CD332 cells were determined using two different methods: (a) a shape-independent electronic sizing technique and (b) a shape-dependent optical image analysis. The cell diameter was measured to be 8.2 6 1.1 mm (mean 6 SD, n 5 1,091,475, the number of donors 5 8) using the electronic sizing technique or 8.7 6 1.2 mm (mean 6 SD, n 5 1508, the number of donors 5 6) by image analysis at initial (isotonic) osmolality, 325 mosm/kg. The cell volume change was measured after the cells were exposed and equilibrated to different anisosmotic conditions. The cell volume was found to be a linear function of the reciprocal of the extracellular osmolality (Boyle van’t Hoff plot) ranging from 163 to 1505 mosm/kg. The volume fraction of intracellular water which is osmotically active was determined to be 79.5% of the cell volume. It was concluded that human CD341CD332 cells osmotically behave as ideal osmometers. This information coupled with cell water and cryoprotectant permeability coefficients as well as their activation energies (to be determined in the ongoing research projects) will be used to design optimum conditions for cryopreservation of human hematopoietic progenitor cells. © 1998 Academic Press Key Words: human hematopoietic progenitors; CD341CD332 cells; osmotic behavior; Boyle van’t Hoff plot.
Autologous bone marrow transplantation permits the use of myeloablative chemotherapeutic regimens in the treatment of cancer. The success of the therapy depends on viable hematopoietic stem cells after cryopreservation. Although successful cryopreservation of bone marrow was reported over 30 years ago (14) and
provides the basis for autologous bone marrow transplantation (4, 12, 17, 36, 40), protocols for cryopreservation have been developed empirically without an understanding of the fundamental cryobiology of human hematopoietic progenitor/stem cells. (It should be noted that Leibo et al. (13) did a careful and excellent investigation on cooling rate dependence of the survival of mouse bone marrow stem cells.) Therefore, questions pertaining to the fundamental cryobiological characteristics of hematopoietic progenitor cells have not been answered. Because the empiric development of cryopreservation techniques has acceptable results when a large volume of bone marrow cells
Received August 1, 1996; accepted October 1, 1997. 1 This work was supported by Methodist Hospital of Indiana, Inc. 2 To whom correspondence should be addressed at Cryobiology Research Institute, Room 204, Potter Building, Hillenbrand Biomedical Engineering Center, Purdue University, West Lafayette, IN 47907. Fax: (765) 494-1193. Email: [email protected]. 40 0011-2240/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.
OSMOTIC BEHAVIOR OF HEMATOPOIETIC PROGENITORS
are processed, the clinical consequences of cryobiology research have been perceived as minor. With new techniques making it possible to isolate small fractions of pluripotent cells, new cryopreservation technology likely will be required, as the proportional injury in the small cell population may result in nonviable transplant preparations. In addition, cryopreservation of purified hematopoietic stem cells is likely to become a key technology in the upcoming era of gene transfer therapy, and optimizing cell cryosurvival rate is critically important. New sources of progenitor cells, such as umbilical cord blood (3), fetal liver (34), and cells generated by ex vivo expansion (44), may require refined protocols to obtain optimal cryosurvival based on the potential difference in their cryobiological characteristics. The major steps used in cryopreservation of cells can be summarized as follows: (i) adding cryoprotective agents (CPAs) to cells before cooling; (ii) spontaneously or artificially inducing ice crystals in the cell suspension at or a few degrees below the freezing point of the cell suspension, and cooling the cells toward a low temperature at which the cells are stored; (iii) warming the cells; and (iv) removing the CPAs from the cells after thawing the sample. Injury to cryopreserved cells has been shown to be caused by each or a combination of the above steps. For example, cells may be damaged by either severe cell volume excursion (osmotic injury) or potential toxicity of the CPAs (8, 21, 37) in steps i and iv. Intracellular ice formation (5–7, 11, 16, 18 –20, 22–25, 29 –31, 35, 39, 43) during the cooling process (in step ii) can kill cells, and the subsequent recrystallization of the intracellular ice during the warming process (in step iii) can also cause or enhance cell injury (25). Based on the previous investigations (8, 21, 23, 25, 30, 32, 42), one needs to know at least the following information to design optimal cryopreservation protocols: (a) cell osmotic characteristics; (b) permeability of the cell to water (Lp) and to the CPAs (PCPA); (c) activation energies (Ea) of Lp and PCPA, respectively;
41
(d) ratio of the cell surface area to volume; (e) intracellular ice formation phenomena; and (f) osmol of solutes initially in the cell. Parameters a– e are cell-type-dependent properties while parameter f is dependent on the concentration and nature of the CPAs. Parameters a– e can be determined from experimental measurements. However, to date, these parameters remain unknown for human hematopoietic progenitor cells. The CD34 antigen, a known progenitor cell marker, is expressed by 1 to 4% of human bone marrow mononuclear cells, while the CD33 antigen, a marker associated with myeloid commitment, is expressed on a higher portion of bone marrow mononuclear cells including a sizable percentage of CD341 cells. CD341CD332 cells include virtually all hematopoietic long-term repopulating cells detected by in vitro assays (1). In the present study, the CD341CD332 cell population isolated from human bone marrow was used as a human hematopoietic progenitor cell model/representative to determine the osmotic characteristics, volume distribution, and osmotically active water volume of the progenitor cells. MATERIALS AND METHODS
Isolation of Human Hematopoietic CD341CD332 Cells Human bone marrow was obtained from 12 healthy donors. Isolated hematopoietic progenitor cells, CD341CD332 cells, were obtained after processing the bone marrow by density gradient centrifugation, soybean agglutinin (SBA), CD341 cell enrichment, and fluorescence-activated cell sorting (1, 3, 4, 17, 40, 41). Leukocyte-rich plasma was obtained after enhanced sedimentation of heparinized bone marrow. This leukocyte-rich product was carefully layered on cushions of Ficoll–Hypaque (density 5 1.077 g/ml) (Sigma, St. Louis, MO). The gradient tubes were then centrifuged at 850g for 30 min and the mononuclear leukocyte layer was aspirated. Mononuclear leukocyte suspensions collected after Ficoll–Hypaque
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were centrifuged and resuspended in Dulbecco’s phosphate-buffered saline without Ca21 or Mg21 (DPBS2) containing 0.5% human gamma globulin (Cohn Fraction II; Sigma) at a concentration of 5 3 106 cells/ml. The cell suspension was placed into AIS MicroCollector T-25 flasks coated with soybean agglutinin (Applied Immune Sciences, Inc., Santa Clara, CA). Each flask contained a total of 2 3 107 mononuclear leukocytes. After a 1-h incubation period at room temperature to ensure sufficient adherence to the flasks, the flasks were gently rocked to dislodge nonadherent cells. These SBA2 cells were aspirated, pooled, centrifuged, and resuspended in DPBS2 containing 0.5% human gamma globulin at a concentration of 5 3 106 cells/ml. For CD341 panning, the cells were collected as described above and placed in the AIS T-25 flasks coated with Murine monoclonal anti-CD341 antibody (clone ICH-3). Following a 1-h incubation, nonadherent cells were removed while the adherent cells were physically dislodged from the AIS T-25 flasks. Cells selected by the AIS technique were typically 20 – 40% positive for the CD341 antigen. A significant and variable portion of the CD341 cells also possessed the CD33 differentiation antigen. These cells were pooled, concentrated by centrifugation, and labeled with two different antibodies for fluorescent analysis and subsequent cell sorting. For cell sorting, cells were labeled with monoclonal anti-CD34 (clone 8G12) (Becton Dickinson, San Jose, CA), directly conjugated with phycoerythrin and anti-CD33 conjugated with fluoroisothiocyanate (Dako, Carpenteria, CA). In these experiments, two different clones of monoclonal anti-CD34 were used in order to optimize the signal of the CD34 antigen by recognizing two different epitopes. The antibodies were simultaneously incubated with the cells for 20 min at 4°C in the dark. Following labeling, the cells were washed once in phosphate-buffered saline (Sigma) containing 1% fetal calf serum (Hyclone, Logan, UT) and 0.1% sodium azide (Sigma). Cells were resuspended in a volume of 3– 4 ml in the wash buffer. The suspension
was placed in the dark at 4°C until further use. Fluorescence-activated cell sorting was accomplished using a FACStar PLUS flow cytometer (Becton Dickson) equipped with a water-cooled argon laser emitting at 488 nm. Subsequent analysis of the cells was performed using Lysis II software (Becton Dickinson). Sorted cells were collected in 12 3 75 mm borosilicate glass tubes (Fisher Scientific, Pittsburg, PA). Typically, the purity of sorted CD341CD332 cells exceeded 97% as determined by the flow cytometer. The purified cells were maintained in M199 medium (Gibco Laboratories, Grand Island, NY) supplemented with 20% (v/v) fetal calf serum at 4°C before treatments and measurements. Preparation of Anisosmotic Cell Suspensions The osmolality of the M199 medium supplemented with 20% (v/v) fetal calf serum was measured to be 325 mosm/kg using an osmometer (Model 3D2; Advanced DigiMatic Osmometer, Needham Heights, MA). This osmolality was used as the initial osmolality of the cells and assumed to be isosmolality. Hyperosmotic solutions were produced by the addition of sucrose to the supplemented M199 medium, and hyposomotic solutions were made by the dilution of the supplemented M199 medium with reagent grade water (Millipore, Burlington, MA). The CD341CD332 cells were exposed to the anisosmotic solutions and allowed to equilibrate with anisosmotic environments for 5 min at room temperature before volume measurement. The osmolality in each final cell suspension was measured. The final osmolalities of anisosmotic cell suspensions were measured to be 163, 243, 270, 325, 585, 925, 1280, and 1505 mosm/kg, respectively. Determination of CD341CD332 Cell Volume Optical image analysis method. Each of the CD341CD332 cell suspensions, with a known osmolality, was placed into 20-mm-deep MicroCell chambers (Conception Technologies, La Jolla, CA). The cells were videotaped using an Olympus BH-2 microscope (Scientific Supply, Schiller Park, IL) with a 20X objective and a
43
OSMOTIC BEHAVIOR OF HEMATOPOIETIC PROGENITORS
video camera (CRYOResources, Ltd., New York, NY) connected to both a Panasonic AG6300 VCR and a video processor image analysis system (Motion Analysis VP320, Santa Rosa, CA). The processing assigned pixels at a manually determined light threshold for an accurate outline of the cell. The software, Expert Vision 2.24X (Motion Analysis), was run on an Amdek System 386 PC for use by the Dynamic Morphology System Software package (DMS, Motion Analysis). Assuming a spherical shape for the CD341CD332 cells, the software was used to generate five still frames of each cell for which an average radial length (the mean length of the radius from the centroid to the boundary over all the pixels used as an outline) was calculated. The volume of standard spherical beads with known diameters (Standard Beads, Coulter Electronics, Ltd., Luton, UK) was measured and used as a control to calibrate the measured geometric dimensions of the CD341CD332 cells. Electronic counter method. The CD341CD332 cell suspensions were placed in the sample chamber of an electronic sizing instrument (Coulter Counter, Model ZM; Coulter Electronics, Ltd.). Using a software package (Average Software, Great Canadian Computer Co., Edmonton, Alberta, Canada), the electronic signal (which is a function of cell volume) of each individual cell that moves through the orifice of the Coulter counter was digitized and analyzed by a microcomputer to determine the cell volume (9, 15, 28). The volumes of over 12,000 CD341CD332 cells per donor were measured using the Coulter Counter. The volume distribution of the cells was determined by using Volume-Software (Great Canadian Computer Co.). The electronic signals of standard spherical beads with known diameters (Coulter Electronics, Ltd.) were measured in a standard isotonic solution, Isoton2 (Fisher Scientific), as a control to calibrate the measured volumes of the CD341CD332 cells. Because the number of CD341CD332 cells from each donor was limited (,500,000 cells per donor sample) and a large number of cells is required for the electronic counter measurement, the electronic
TABLE 1 Geometric Dimensions of Human Hematopoietic CD341CD332 Cells at 325 mosm/kg Diameter Volume Surface (mean 6 SD) (mean) area (mean) Number of mm mm3 mm2 cells Method 8.2 6 1.1 8.7 6 1.2 a b
289 345
211 238
1,091,475a Coulter 1,508b Optical
Obtained from 8 different donors. Obtained from 6 different donors.
counter measurement was conducted only at 925, 325, and 243 mosm/kg, respectively. Ideal Osmometer Hypothesis It was hypothesized that the CD341CD332 cells behaved as ideal osmometers, which means the cell volume changes linearly as a function of the reciprocal of the osmolality of the external solution, i.e., the Boyle van’t Hoff (BVH) relationship (2). The mathematical expression of the BVH relationship is V/V i 5 ~1 2 V b /V i !M i /M 1 V b /V i , where V is the cell volume at osmolality M, Vi is the cell volume at isosmolality Mi (325 mOsm in this study), and Vb is the osmotically inactive cell volume (including osmotically inactive intracellular water and nonaqueous/solid part of the cells). Statistical Analysis Experimental data were analyzed and expressed as means 6 SD. Linear regression analysis was used to analyze the Boyle van’t Hoff plot. A Student’s t test was used to examine the difference between the human hematopoietic progenitor cell mean volumes measured by the two different methods (i.e., the optical and Coulter Counter methods) assuming that the data population follows a normal distribution (27). RESULTS
Geometric dimensions (diameter, volume, and membrane surface area) of the human
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CD341CD332 cells at 325 mosm/kg were determined using the optical image analysis and Coulter Counter methods (Table 1). In the optical method, cell diameter was measured, and the cell volume and surface area were calculated assuming the sphericity of cells. In the Coulter technique, cell volume was measured and cell diameter and surface area were calculated, again assuming the sphericity of cells. The optical image analysis showed that the CD341CD332 cells in the osmotic range 136 to 1505 mosm/kg maintained a spherical shape. The mean diameter of all cells measured by optical image analysis (8.7 6 1.2 mm) was consistent with that determined by the Coulter Counter technique (8.2 6 1.1 mm). There was no statistical difference between the two mean values (P . 0.5). In addition, the mean value of the cell diameter was donor-independent (P . 0.5). Although the resolution of the equipment used and the assumption of the spherical shape of the cells (in the optical imaging method) might create some errors in measurement, the results obtained using the different methods showed a similar volume distribution pattern for the CD341CD332 cell population. Figure 1 shows cell volume distributions of six samples, obtained from six different donors, determined by the Coulter Counter technique. Figure 2 shows a similar pattern of cell volume distribution determined by optical image analysis (the number of donors 5 6). In the Coulter Counter measurement, it was assumed that any particle with a diameter less than 6 mm (113 mm3 in volume) was debris and any particle with a diameter greater than 10 mm (523 mm3 in volume) was a cell aggregate (i.e., more than two cells moving simultaneously through the orifice in the Coulter Counter device). These particles, which consist of 0.1% of the total cells selected, were excluded in determining the dimensions of the CD341CD332 cells. The CD341CD332 cell volumes (relative to isotonic cell volume) versus the reciprocal of the external osmolality (i.e., Boyle van’t Hoff plot) are shown in Fig. 3. A linear fit (V/Vi 5 0.886 (325/M) 1 0.205; r2 5 0.98) was obtained using a least-square fit method (Sig-
maPlot, Jandel Scientific Corp., San Rafael, CA). The linearity of the Boyle van’t Hoff plot indicates that the CD341CD332 cells act as ideal osmometers in the range of 163 to 1505 mosm/kg. The nonzero (V/Vi) axis intercept of 0.205 indicates that 20.5% of the cell volume is osmotically inactive. DISCUSSION
The optical image analysis is one of the most commonly used approaches in measuring cell dimensions. This technique can be used to measure the static cell volume or the kinetic volume change of given individual cells. Only a few cells are required for this technique. However, cell shape dependence and time consumption in the image analysis as well as limitation of the optical resolution are disadvantages of the optical image method. The electronic counter technique used in this study is a cell-shapeindependent approach and has been shown to be a very reliable tool to measure the static volume distribution and kinetic volume change of a large number of cells regardless of spheric shape (9, 15, 28). Results generated from both techniques agree well with each other. From this study, it is known that the mean volume of the human hemetopoietic progenitor cells is approximately 289 (by the optical method) or 345 mm3 (by the Coulter Counter method), as indicated in Table 1. These values are comparable with the human lymphocyte mean volume, 290 mm3, as determined by using Coulter Counter (10). The present study also indicates that 20.5% of the human hematopoietic cell volume is osmotically inactive (or, in other words, 79.5% of the cell volume is osmotically active water), which is lower than that (32– 36%) of the human lymphocytes (10, 13). Based on the Boyle van’t Hoff plot, it is concluded that human CD341CD332 cells osmotically behave as ideal osmometers. The information obtained from the present study concerning the CD341CD332 cell volume distribution, cell osmotically inactive volume, and osmotic characteristics provides a basis for ongoing investigations to determine the hematopoietic progenitor cell water and cryoprotectant permeability co-
OSMOTIC BEHAVIOR OF HEMATOPOIETIC PROGENITORS
FIG. 1. Coulter Counter-determined volume distributions of six human hematopoietic CD341CD332 cell samples obtained from six different donors (n 5 6).
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FIG. 2. Volume distribution of human hematopoietic CD341CD332 cells determined by optical image analysis (number of cells measured 5 1508 cells from six donors).
FIG. 3. The CD341CD332 cell volume (relative to isotonic cell volume) versus the reciprocal of the extracellular osmolality (Boyle van’t Hoff plot). V expresses the total cell volume at osmolality M; Vi is the cell volume at isosmolality Mi (325 mosm/kg). Two data points indicated by arrows were determined by the Coulter Counter method. The other data points were determined by using optical image analysis. A linear fit (V/Vi 5 0.87(325/M) 1 0.21; r2 5 0.97) was obtained using a least-square fit method.
OSMOTIC BEHAVIOR OF HEMATOPOIETIC PROGENITORS
efficients and their activation energies. These cell biophysical parameters are required and essential to lead to a better understanding of the fundamental cryobiology of the progenitor cells and to permit the development of optimum procedures for cryopreservation.
10.
11.
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