Stem Cells, Vol. 19, No. 3, 212-218,
May 2001
© 2001 AlphaMed Press
An Efficient Method for the Cryopreservation of Fetal Human Liver Hematopoeitic Progenitor Cells
Jiun Zhaoa,
Hsiao-Nan Haoa,b,
Ronald L. Thomasa,
William D. Lymana,b,c
a Children's Research Center of Michigan; Departments of
b Pediatrics and
c Psychiatry and Behavioral Neurosciences, Wayne State University School of Medicine, Detroit, Michigan, USA
Key Words. Cryopreservation • Fetal human liver • Hematopoietic progenitor cells • Stem cells
Jiun Zhao, M.D., 3901 Beaubien, Detroit, Michigan 48201, USA. Telephone: 313-745-2400; Fax: 313-745-0282.
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ABSTRACT
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The use of human hematopoietic progenitor cells (HPC) for transplantation requires efficient recovery methods and cryopreservation procedures. The purpose of this study was to determine cryopreservation techniques for fetal human liver (FHL) CD34+ cells. We assessed FHL HPC recovery efficiency after freezing and thawing by viability testing, fluorescence-activated cell sorting analysis, and colony-forming ability under different conditions. We also determined optimal cell freezing concentrations and the effect of rate-controlled freezing on cell recovery. Lastly, cell recovery after varying freezing time periods was examined. Our results indicated that optimal cell recovery occurs when: A) cryopreservation medium consists of either 5% dimethylsulphoxide (DMSO) or 10% DMSO in combination with either 20% fetal bovine serum (FBS) or 70% FBS and when Iscove's modified Dulbecco's medium consists of not more than 10% DMSO; B) a rate-controlled freezing device container is used; C) CD34+ cells are frozen at a concentration of 1 x 106/ml, and D) a thawing temperature of 37°C is used. These observations indicate that cryopreservation of FHL HPC is possible for up to 18 months in optimal conditions without losing hematopoietic activity.
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INTRODUCTION
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Hematopoietic progenitor cell (HPC) transplantation is becoming an important therapeutic strategy for the treatment of cancer, congenital immunodeficiencies, inborn errors of metabolism, and various hematological diseases. Finding appropriate bone marrow donors for these patients, however, continues to be a major problem as it complicates positive therapeutic outcomes [1]. In order to overcome this limitation, attempts are being made to find alternative sources of HPCs. A principal alternative may be fetal human liver (FHL)-derived HPCs. Improved results are anticipated because this tissue type is a rich resource, the cells can be purified to a relatively homogeneous population and they have considerable proliferative potential. Recently, many experimental data have shown that these cells have decreased graft-versus-host potential [2, 3].
The widespread use of various treatment protocols utilizing HPCs have resulted in an increased demand for cryobiological techniques for storing these cells. This need has led to a reexamination of many cryobiological practices. Much progress has been made in bone marrow, umbilical cord blood, and peripheral blood cryopreservation techniques [4-6]. However, a systematic cryopreservation study for FHL-derived HPCs has not been described. Muench et al. reported that FHL-derived HPCs differ from other sources of progenitor cells [2, 7]. Huang et al. claimed that FHL-derived HPCs are the best target for genetic manipulation [8-10].
The aim of this study was to optimize the cryopreservation of FHL HPCs based on recently described cryopreservation techniques [4-6]. Specifically, we investigated various combinations of dimethylsulfoxide (DMSO) and fetal bovine serum (FBS) concentrations in the freezing medium, various thawing temperatures, control of the cooling rate, cell freezing concentration, and long-term freezing on cell recovery.
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MATERIALS AND METHODS
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FHL Tissues and CD34+ Cell Preparation
The present study is part of an ongoing research protocol that has been approved by the Wayne State University School of Medicine Human Investigations Committee. FHL was collected after elective termination of intrauterine pregnancies from otherwise normal healthy women. Informed consent was obtained from all participants. The tissues investigated in this study were obtained from human fetuses ranging in age from 16 to 24 weeks.
Dissociated liver cell suspensions were prepared by passing tissue samples through stainless steel grids in Iscove's modified Dulbecco's medium (IMDM; GIBCO; Grand Island, NY). Connective tissue was separated from the cell suspension by gravity. The mononuclear cells were separated by centrifugation through a discontinuous density gradient with Ficoll-Hypaque (Pharmacia Biotech; Uppsala, Sweden; http://www.pnu.com). The cells in the low-density layer were collected and washed with phosphate buffered saline Ca2+/Mg2+ free (GIBCO), supplemented with 2% FBS. The red blood cells were lysed with an ammonium chloride solution (Stem Cell Technologies Inc.; Vancouver, BC, Canada; http://www.stemcell.com). CD34+ cells were isolated using a MiniMACS CD34 progenitor cell isolation kit (Miltenyi BioTec; Auburn, CA; http://www.miltenyibiotec.com) as described by Choi [11].
Cryopreservation Procedures
Recovery efficacy was evaluated using five separate methodological approaches to cryopreservation: A) FBS and DMSO concentrations were varied using six combinations of DMSO (Sigma; St. Louis, MO; http://www.sigma-aldrich.com) and FBS (GIBCO) in IMDM (70% FBS + 5% DMSO, 70% FBS + 10% DMSO, 70% FBS + 20% DMSO, 20% FBS + 5% DMSO, 20% FBS + 10% DMSO, and 20% FBS + 20% DMSO); B) throwing temperature conditions were varied using progressively higher temperatures (22°C, 37°C, or 42°C); C) freezing technique was varied by placing cells either into a "Nalgene Cryo 10 C" freezing container (Nalgene; Rochester, NY) or by directly placing them in a freezer overnight, with the temperature at –70°C. Each sample was then transferred to a liquid nitrogen tank the following day; D) freezing time period was varied by using five different time periods (1, 6, 12, and 18 months) of post-cryopreservated CD34+ cells, and lastly, E) cell freezing concentrations were examined at varying milliliters (1, 2.5, 5, and 10 x 106/ml). For all experiments, except experiment 5 which varied cell concentrations, a single concentration of 1 x 106/ml of CD34+ cells was applied. Time of freezing duration was also set at 3 months, with the exclusion of experiment 4 which varied the freezing time points. One ml of cell suspension was frozen in 2-ml sterile, thermo-stable, plastic vials (Fisher; Itasca, IL). When thawing cells, freezing vials were kept at 37°C, until ice crystals disappeared. The volume was slowly increased to 10 times the initial volume with IMDM containing 2% FBS, and a small aliquot of cell was then taken for cell count. Using a 1:1 dilution of 0.4% trypan blue (GIBCO), cell viability was then examined. Cells excluding the dye were counted both before freezing and after thawing, and the absolute number was calculated to determine cell recovery. Cells were then washed for subsequent experiments. All five experiments were repeated three times each, and cell recovery values obtained were then averaged.
Cell Phenotype Analysis
Cellular phenotype and viability were analyzed by fluorescence-activated cell sorting (FACS) using standard procedures. Briefly, before and after cryopreservation, column-purified CD34+ cells were labeled with fluorescein isothiocyanate (FITC)-conjugated anti-CD3 monoclonal antibody and phycoerythrin (PE)-conjugated anti-CD34 monoclonal antibody (Beckman Coulter, Inc.; Brea, CA; http://www.coulter.com). FITC- and PE-conjugated mouse IgG monoclonal isotypes were used as negative controls. Flow cytometry was performed on a FACS vantage COULTER EPICS XL-MCL.
Colony Assay
Colony assay was performed in methylcellulose medium with recombinant cytokines (H4435, Stem Cell Technologies, Inc.) as described by Hogge [12]. After assessing cell viability, 1 x 103 cells were seeded in triplicate in 35 mm2 plates with 1 ml of methylcellulose medium. Plates were then incubated in a humidified atmosphere of 5% CO2 in air at 37°C for 14 days. BFU-E and colony-forming unit-granulocyte, macrophage were examined. Clones present with greater than 40 cells were considered as colonies. The percentage of colony recovery was calculated by using the number of colonies found after freezing and dividing this quantity by the number of colonies that existed before freezing. The final quantity was then multiplied by 100.
Statistical Analysis
One-way analysis-of-variance (ANOVA) procedures were employed to assess the effect of average percent differences in cell recovery over different experimental methodologies. The assumptions for the proper application of ANOVA were verified in each analysis. Levene's test for homogeneity of variance was verified in the output. Post-hoc examinations were conducted using Sidak's test. Statistical significance was achieved in pair-wise comparisons at a p 
0.05, two-tailed. An independent samples t-test was employed to compare average percent differences in cell recovery in the rate versus non-rated controlled experiment. Again, appropriate assumptions were checked and verified. Statistical significance was achieved with a p 0.05, two-tailed.
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RESULTS
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Cryopreserving FHL CD34+ Cells
Effects on cell recovery as a result of varying concentration percentages of both FBS and DMSO are shown in Figure 1
. The average percent of cell recovery obtained was highest using the concentration combination of 70% FBS + 10% DMSO (93.3 + 7.2), although concentration combinations 70% FBS + 5% DMSO (92.0 ± 2.65) and 20% FBS + 5% DMSO (92.3 ± 3.1) reported extremely similar mean results as well as lower standard deviations. Cell recovery was lowest using 70% FBS + 20% DMSO (76.7 ± 10.7). One-way ANOVA revealed that the largest mean differences in average percent cell recovery, among the six concentration combinations, occurred between concentration combinations 70% FBS + 10% DMSO and 70% FBS + 20% DMSO (16.7 ± 5.3; p = 0.12); 20% FBS + 5% DMSO and 70% FBS + 20% DMSO (15.7 ± 5.3; p = 0.17); and 70% FBS + 5% DMSO and 70% FBS + 20% DMSO (15.3 ± 5.3; p = 0.19).
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Figure 1. Effects of DMSO and FBS combination concentration in cryoprotectant solution on FHL CD34+ cell recovery. The number of the viable cells from each indicated cryoprotectant solution-treated culture was estimated as described in Materials and Methods. Data are expressed relative to values measured in total cell count. Data are mean ± standard deviation (bars) values of triplicate samples from three or more independent experiments.
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Results of thawing temperature on average percent of progenitor cell recovery are shown in Figure 2A. The highest average percent cell recovery occurred at 37°C (94.3 ± 5.7) compared with 22°C and 42°C. The average percentages of CD34+ cell recovery between rate versus non-rate controlled experiments are shown in Figure 2B. Mean cell recovery was 95 ± 2 in the rate-controlled group and 93 ± 2.65 in the non-rated controlled group, statistically insignificant (p = 0.36).
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Figure 2. Effect of thawing temperature, cryopresevation procedures, freezing time period, and cell freezing concentration on FHL CD34+ cell recovery. A) Cells were immediately defrosted from 20% FBS and 5% DMSO cryoprotectant solution at 22°C, 37°C, and 42°C. Cell viability was scored as described in Materials and Methods. B) Cells were frozen by either cryopresevation rate control procedure or non-cryopresevation rate control procedure, and stored in liquid nitrogen for 3 months. C) Cells were stored with 20% FBS and 5% DMSO cryoprotectant solution for 1, 6, 12, and 18 months and immediately defrosted at 37°C. D) Isolated CD34+ cells were resuspended in the 20% FBS with 5% DMSO cryoprotectant solution at different concentrations (1, 2.5, 5, and 10 x 106/ml) and stored at –180° C for 3 months. Cells were defrosted in a water bath at 37°C and cultured in IMDM with 10% FBS. Cell viability from each sample was examined using trypan-blue exclusion. Data are expressed relative to values measured in total cell count. Data are mean ± standard deviation (bars) values of triplicate samples from three independent experiments.
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Average percent differences in cell recovery, using a concentration of 1 x 106/ml were examined by varying freezing periods (1, 6, 12, and 18 months). One-way ANOVA did not reveal any statistically significant differences among the four different freezing periods (p = 0.99). Average percent cell recovery remained strikingly similar at each time period, with the approximate mean equal to 83.2 ± 6.5. Figure 2C
displays these results graphically.
Four different freezing cell concentrations (1, 2.5, 5, and 10 x 106/ml) were also evaluated and compared for their respective efficacy in cell recovery. Mean cell recovery with trypan blue exclusion was highest in concentrations 1 x 106/ml (91.3 ± 1.5) and 2.5 x 106/ml (92 ± 3). Although each produced nearly identical means, the obtained standard deviation was twice as low in concentration 1 x 106/ml. Percentage of cell recovery was decreased using concentration 10 x 106/ml. One-way ANOVA did show that a statistically significant difference existed among the four different cell concentrations in average percent cell recovery (p = 0.002). The concentration using 10 x 106/ml differed significantly when compared against concentration 1 x 106/ml (11.3 ± 2.2; p = 0.004) and concentration 2.5 x 106/ml (12 ± 2; p = 0.003) (Fig. 2D).
FACS Analysis Cell Phenotypes of Pre- and Post-Cryopreserved Column-Purified CD34+ Cells
The purity of column-purified CD34+ FHL cells was examined using anti-CD4 and anti-CD34 antibodies. The population of CD34+ cell was over 95% after liver cells were purified with the magnet bead CD34 purification column (Fig. 3B). Comparing the freshly column-purified cells with those 18-month cryopreserved cells, the cell phenotype of pre- and post-cryopreserved cells remained the same (Fig. 3B and C). This result indicates that column-purified CD34+ cells passed through this cryopreserved procedure without losing and changing their cell markers.
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Figure 3. Flow cytometric profiles of column isolated CD34+ cell. The phenotype of column purified pre- and post-cryopreserved cells was estimated using anti-CD3 and CD34 antibodies. Panel A: isotype negative controls were used to gate out the nonspecific stained cells. Panels B and C indicate that the great majority of column-purified CD34+ cells (>97%) were labeled by FITC before and after cryopreservation for 18 months, respectively.
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Cell Clonogenic Assay
Average colony numbers recovered by varying cell concentrations (1, 2.5, 5, and 10 x 106/ml) revealed that higher average counts were obtained using concentrations 1 x 106/ml (38.2 ± 22.3) and 2.5 x 106/ml (38.9 ± 25.9). Concentration 10 x 106/ml (28.1 ± 13.0) recovered the lowest average number of colonies. However, the standard deviations calculated for each of the four concentrations examined indicated wide variability. Colony numbers for concentration 1 x 106/ml ranged from 10.0 to 71.0; concentration 2.5 x 106/ml from 16.0 to 98.0; concentration 5 x 106/ml from 23.0 to 53.0; and concentration 10 x 106/ml from 0.0 to 42.0 (Fig. 4).
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Figure 4. Comparison of the colony formation ability of both fresh isolated and thawed FHL CD34+ cells after cyropreservation stored for 18 months. After cells were cultured in methylcellulose medium with growth factors for 15 days, granulocyte-macrophage progenitor cell-derived colonies (CFU-GM) and erythroid progenitor cell-derived colonies (BFU-E) were assessed. Upper and lower panels: bright field photomicrograph of CFU-GM and BFU-E colonies from fresh isolated CD34 cell (A) and thawed FHL CD34+ cells cyropreserved stored for 18 months (B). Original magnification x 40.
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DISCUSSION
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In this study, we investigated the efficacy of five separate methodologies and their respective efficacy on cell recovery of cryopreserved FHL-derived HPCs. The most commonly used cryoprotectant, DMSO, penetrates cells rapidly and avoids intracellular freezing by preventing a substantial increase in the external osmotic pressure and reducing ice crystal formation. The effectiveness of DMSO generally increases with its concentration, but it can also have deleterious osmotic and toxic effects on these cells [4]. Our data (Fig. 1) indicated that although 10% DMSO in combination with 70% FBS produced the highest average percent cell recovery (93.3 ± 7.2), such concentrations in IMDM are not clearly optimal over others to protect the cell membrane and maintain the freezing medium pH balance.
Similar recovery results were obtained using a combination of 5% DMSO in combination with either 70% or 20% FBS. Rapid thawing, where frozen cells are warmed at a rate more than 90-100°C per min, is usually recommended to protect cryopreserved cells from mechanical destruction by intracellular recrystallization or from cell expansion by dilution shock [5]. Katayama et al. [13] reported that hematopoietic activity of the HPCs is unaffected when they are infused slowly within 60 min, or even when they are thawed gradually at room temperature. Our data indicated that there was no significant difference in cell recovery for various thawing temperatures investigated (22°C, 37°C, and 42°C). However, thawing cells at 37°C resulted in a higher average yield. It is suggested that rapid thawing up to the physiological threshold is optimal for these cells.
Frozen cells are injured by the direct effects of low temperatures and by formation of ice crystals. Intracellular ice formation coupled with high rates of cooling may rupture the cell, and extracellular ice formation results in increased extracellular medium osmolality as water is taken up, causing severe dehydration [6]. Rate-controlled cryopreservation reduces this potential damage. Although our results indicated that both techniques performed equally well, given the potential for severe dehydration in non-rate-controlled freezing, rate-controlled freezing should be continued.
Freezing unsorted FHL Ficoll-Hypaque-fractionated cells at high concentration can simplify the cryopreservation procedures. However, due to tissue condition variability and frequent cell clumping which occurred during thawing, a constant yield was hard to obtain, therefore FHL CD34+ cells were studied. We evaluated the concentration at 1. 2.5, 5, and 10 x 106/ml. Our data suggest that the higher the freezing concentration, the higher the degree of cell clumping that was observed. The clumped cells were associated with reduced cell viability, despite the addition of DNase (data not shown). It had been reported that when a clump is observed, it is usually composed of 90%-100% dead cells [14]. Our viability counts did not include clumps. To minimize diluting shock and cell clumping during thawing, a CD34+ cell-freezing concentration of 1 x 106/ml is recommended. Makino et al. reported that if the freezing procedure is optimal and no cell damage has occurred, no further damage would be expected during the storage period [5]. For long-term cryopreservation, however, it is necessary for the storage temperature to be low enough to block all enzymatic pathways and metabolism of the cells. Our trypan-blue exclusion and colony-forming assay indicated no statistically significant difference in mean percent cell recovery after cryopreservation in liquid N2 vapor, between monthly periods of either 1, 6, 12, or 18 months. In traditional clonogenic assays, such as the BFU-E and CFU-GM assays, it is not possible to express and compare a total cell recovery due to the extreme variability of the colony appearance. Moreover, inter-laboratory assay comparisons are difficult owing to variations in methodology. We used clonogenic assays only to check the cell colony formation ability. After trypan blue exclusion, a constant number of cells were used. All our experiments were checked with colony formation assay. Our results indicate that after trypan blue exclusion, the cell colony-forming ability was not affected.
In summary, an efficient method for the cryopreservation of FHL-derived HPCs was determined. Our recommendations based on this study are as follows: A) a cryopreservation medium consisting of either 5% DMSO or 10% DMSO in combination with either 20% FBS or 70% FBS and in IMDM, not to exceed 10% DMSO; B) using a rate-controlled freezing device container; C) freezing CD34+ cells at a concentration of 1 x 106/ml, and D) utilizing a thawing temperature of 37°C. These results provide efficient methodological information for the isolation and storage of fetal human liver hematopoietic stem cells that may be useful in clinical transplantation trials.
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ACKNOWLEDGMENTS
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We thank Ms. Nancy Fine and Mr. Steve Buck for their expertise in flow cytometry analysis and Dr. Joseph Kaplan for his valuable suggestions during preparation of the manuscript.
Support for this study was provided by the Children's Research Center of Michigan.
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References
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Received January 8, 2001;
accepted for publication February 2, 2001.
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Abstract
Introduction
