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Cryopreservation Does Not Affect Proliferation and Multipotency of Murine Neural Precursor Cells

^a Department of Neurology, University of Leipzig, Leipzig, Germany;
^b Department of Neurology, Technical University of Dresden, Dresden, Germany

Key Words. Neural precursor cells • Cryopreservation • Apoptosis • Proliferation

Correspondence: Javorina Milosevic, Ph.D., Department of Neurology, Max-Bürger-Forschungszentrum, Johannisallee 30, 04103 Leipzig, Germany. Telephone: 49-341-9725874; Fax: 49-341-9725878; e-mail: javorina.milosevic{at}medizin.uni-leipzig.de

	ABSTRACT

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Stem cell research offers unique opportunities for developing new medical therapies for devastating diseases and a new way to explore fundamental questions of biology. Establishing an efficient freezing protocol for neural precursor cells (NPCs) is of great importance for advances in cell-based therapies. We used fluorescence-activated cell sorter–based cell death/survival analysis and Western blot analysis of proliferation markers (proliferating cell nuclear antigen) and prosurvival proteins (Bcl-2) to study the effect of a variety of cryoprotective agents on fetal mouse forebrain NPCs. Neurospheres frozen at –70°C or in liquid nitrogen in a rate-controlled manner and thawed after 5 days retained viability of 60%–70% measured 24 hours after thawing. However, 1 week after thawing, viability dropped to 50%–60%. Using a clonogenic sphere formation assay, we showed that recovery rate of frozen NPCs was approximately 26% and did not significantly differ between dimethyl sulfoxide (DMSO)– and glycerol-supplemented samples. Application of the caspase inhibitor zVAD-fmk during freezing or in the first week after thawing resulted in protection of cryopreserved neurospheres after thawing but not during the freezing process, indicating that apoptosis limits recovery of NPCs. Cell survival was not reduced in cells that were enzymatically separated before cryopreservation. Optimal protection of NPCs was achieved when 10% DMSO alone or in a combination with 10% fetal calf serum (FCS) was used. However, 10% glycerol alone was equally effective. Using these protocols, NPCs retained their multipotency and differentiated into both glial (GFAP-positive) and neuronal (Tuj1-positive) cells. Percentage of Tuj1-positive cells in 5% and 10% DMSO, in 10% DMSO + 10% FCS, and in 10% glycerol remained at the same level as before freezing and varied from 5%–7%. We conclude that cryopreservation (up to 1 month at –70°C and up to 1 year in liquid nitrogen) does not markedly alter the rate of proliferation and multipotency of murine neural precursor cells.

	INTRODUCTION

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Stem cells are "master cells" that give rise to specific cells in the body. From a developmental standpoint, murine neural stem cells represent an accessible and important system for studies of basic stem cell properties such as self-renewal and multipotency [1]. The clinical implications of neural stem cells are potentially profound [2–4]. Cryopreservation may be a prerequisite for quality assurance, storage, and distribution required for tissue that shall be used clinically. Therefore, development of appropriate cryopreservation techniques is required. Cryopreservation of murine neural precursor cells (NPCs) has been done using dimethyl sulfoxide (DMSO) (Me₂SO) [5, 6]. However, the role of different cryoprotectants in the preservation of murine NPCs has not been studied in detail.

Many factors alter the effectiveness of cryopreservation of eukaryotic cells, including cell type and size, growth phase and rate, growth medium composition, incubation temperature, pH, cell water content, lipid content and composition of the cells, density at freezing, composition of the freezing medium, cooling rate, storage temperature and duration of storage, warming rate, and recovery medium [7, 8]. Recently, in hematopoietic stem cells (HSCs) and other cell types, the involvement of apoptosis in cryoinjury has been proposed [9–11]. However, one of the most important factors in cryoprotection is the composition of the medium used for freezing. Various approaches for long-term storage and preservation of biological material are based on different cryoprotective agents such as DMSO, ethylene glycol, glycerol, and sugars. Among other sugars, natural disaccharide trehalose, found in high concentrations in organisms tolerant to desiccation and extremely low temperatures, has become interesting because of its extraordinary capability to preserve structural integrity of frozen cells [12, 13]. Trehalose is effective in cryopreservation, being present either exogenously or endogenously [14–16].

We believe that neural stem cells will be used clinically. Therefore, developing a cryopreservation medium with little toxicity is mandatory. In addition, serum, which is often used in cryopreservation protocols, must not be applied to cells aimed at clinical use. DMSO, which is often used to protect cells during freezing and thawing, is rather toxic. Therefore we studied another quickly penetrating protectant, ethylene glycol. In addition, we evaluated slowly penetrating (glycerol) or nonpenetrating (disaccharide trehalose) cryoprotective additives as an alternative to DMSO. The effect of additional serum was investigated with all cryoprotectants. To test the notion of apoptosis involvement during freezing process and in post-thawing recovery of NPCs, caspase inhibitors were used as a supplement to the serum-free medium in combinations with various cryoprotectants.

	MATERIALS AND METHODS

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Neurosphere Isolation and Propagation
Forebrain tissue was dissected and pulled from C57/Bl6 E.16 mouse embryos and expanded in culture as described previously [5, 17]. Pregnant females were euthanized according to NIH guidelines and the approval of the local animal care committee. Dissected tissue samples were incubated in 0.1 mg/ml papain solution (Roche, Basel, Switzerland) for 30 minutes at room temperature, incubated in antipain (50 µg/ml; Roche) for 10 minutes at 37°C, and finally homogenized by gentle triturating using fire-polished Pasteur pipette. The cells were added to plastic six-well plates (Costar, Bodenheim, Germany) and maintained in serum-free media for expansion comprising Dulbecco’s modified Eagle’s medium (high glucose)/F-12 mixture (1:1), B27 supplement (1:50), 20 ng/ml of human recombinant epidermal growth factor (EGF) (PromoCell, Heidelberg, Germany), and 20 ng/ml basic fibroblast growth factor (PromoCell), 100 U/ml penicillin, and 100 µg/ml streptomycin (PAA Laboratories, Pasching, Austria). The fresh growth factors were supplemented every fifth day, allowing cells to grow into floating neurospheres. To ensure better insight into the cell number before and after the freezing process, the cells were grown in a monolayer via plating on polyornithine-fibronectin–precoated dishes in a density of 10,000 cells per cm². The cells were cultured at 37°C in 95% air (21% O₂) and 5% CO₂ in a humidified chamber.

Cryopreservation Techniques and Post-Thawed Propagation of NPCs
Two-week-old murine neurospheres were frozen within 24 hours using a rate-controlled freezing system (Cryo Freezing Container, Nalgene, Rochester, NY) with a cooling rate of 1°C per minute. For freezing, serum-free expansion medium was used, but it was supplemented with different cryoprotective additives (DMSO, glycerol, trehalose, ethylene glycol), with or without 10% fetal calf serum (FCS) and caspase inhibitor zVAD-fmk (Enzyme Systems, Livermore, CA). Some of the samples were enzymatically treated with Accutase (PAA Laboratories) for 15 minutes at 37°C, allowing cell number determination. Approximately 2 to 5 x 10⁶ cells per cryogenic vial (Nalgene) were frozen. The samples were stored in a freezer at –70°C, and after 24 hours the parallel samples were transferred to liquid nitrogen (–186°C). Five days after freezing, from both –70°C and liquid nitrogen, the cells were thawed rapidly in a 37°C water bath with continuous agitation and were diluted 1:10 with the culture medium. Four hours later, freezing medium was replaced with a serum-free expansion medium, and neurospheres were maintained as described above. To prevent large sphere formation, neurospheres were treated as usual, twice a week with Accutase for 15 minutes at 37°C. Caspase inhibitor zVAD-fmk (20–80 µM) was supplemented during post-thawing recovery phase for 2 or 5 days.

Measurement of Cell Viability and Cell Death
To measure trypan blue exclusion, fresh or frozen-thawed NPCs were incubated in triplicates in a 0.25% dye solution (Gibco, Carlsbad, CA). The number of total and trypan blue–positive cells was then determined by counting at least 200 cells per sample in a hemocytometer. Cell viability (percent) means the ratio of the number of trypan blue–impermeable cells in total cell counts (trypan blue–impermeable cell number/total cell number). Necrotic cells (percent) referred to percentage of trypan blue–positive cells.

For apoptosis quantification, NPCs (1 x 10⁶) were harvested 24 hours after thawing and washed three times with phosphate-buffered saline (PBS) (pH, 7.2). Early apoptosis was analyzed using an annexin-V assay kit from BD Pharmingen (San Jose, CA). Data were analyzed by the Cell Quest program (BD Pharmingen). DNA content/cell cycle analysis and the cell death measurement were performed by detecting propidium iodide (PI)–stained NPC nuclei as described [18]. The apoptotic cells with degraded DNA (late apoptosis) appear as cells with hypodiploid DNA content and are represented in so-called sub-G1 peaks on DNA histograms [19]. Twenty-four hours or 7 days after thawing, the nuclei were prepared by lysing 1 x 10⁵ cells in 250 µl of hypotonic lysis buffer (0.1% sodium citrate, 0.1% Triton X-100, and 50 µg/ml PI) and were subsequently analyzed by flow cytometry using a FAC-Scan (Becton, Dickinson, Heidelberg, Germany) and Cell Quest analysis software. The sample flow rate during analysis did not exceed 500 to 600 cells per second. At least three measurements were performed for each sample.

Clonogenic Survival Assay
NPC survival after exposure to freezing process in 10% DMSO and 10% glycerol was measured via a colony-forming assay. Clonogenicity was determined by measuring colony formation as described [20]. In brief, after the cells were thawed and washed, the stem cells were adjusted to a final concentration of 5 x 10⁴ per milliliter in 9-ml culture medium. The cells were seeded in triplicates in six-well plates. Relative survival value was calculated by determining the ratio of colony-forming units (CFUs) (secondary neurospheres) after cryoprotectant exposure to those in fresh samples after 14 days of incubation at 37°C in a humidified 5% CO₂ atmosphere. Secondary neurospheres were scored using an inverted microscope applying standard criteria for their identification.

Neurosphere Differentiation
Cells were differentiated by plating them onto poly-L-lysine–coated 24-well plates at the density of 50,000 cells per well. To induce differentiation, Neurobasal medium (Gibco, Carlsbad, CA) was supplemented with 1% FCS, interleukin-1b (Sigma, St. Louis) (100 pg/ml), and 5 µM forskolin (Sigma). The cells were allowed to differentiate for up to 7 days at 37°C in a humidified atmosphere in 5% CO₂, 95% air. The differentiation medium was changed after the third day.

Immunocytochemistry
Differentiated cells were fixed in 4% paraformaldehyde in PBS for 15 minutes at room temperature and washed with PBS. The cells were then counterstained with the DNA-binding dye 4'-6'-diamidino-2-phenylindole (DAPI) (2 µg/ml in PBS) for 15 minutes at room temperature, twice washed in PBS followed by incubation in blocking buffer (10% FCS; 0.2% Triton-X 100 in PBS; pH, 7.2) for 30 minutes at room temperature. After incubation with a primary antibody (1 hour at room temperature in blocking buffer), the cells were incubated with biotin-conjugated secondary antibodies followed by incubation with streptavidine Alexa Fluor 488 conjugate or Alexa Fluor 594 conjugate (Molecular Probes, Eugene, OR). Coverslips were mounted onto glass slides and examined under a fluorescence microscope (Zeiss Axiovert 200). Acquisition of the stained cells was performed using the image-analysis software AxioVision 4 (Zeiss, Jena, Germany).

The following antibodies were used for immunocytochemistry: rabbit polyclonal anti-ß-tubulin III (anti-Tuj1) antibody (Covance, Princeton, NJ); mouse monoclonal anti-glial fibrillary acidic protein (anti-GFAP antibody [Chemicon, Temecula, CA]); mouse monoclonal anti-oligodendrocyte marker O4 (Chemicon); biotin-conjugated secondary antibodies (Molecular Probes); streptavidin, Alexa Fluor 488 conjugate; and streptavidin, Alexa Fluor 594 conjugate (Molecular Probes).

Immunoblotting
Two weeks after thawing, NPCs were collected by mild centrifugation (x 100g for 5 minutes), washed with ice-cold PBS, and lysed in buffer (10 mM HEPES-KOH [pH, 7.9], 10 mM KCl, 1.5 mM MgCl₂, 0.1% NP-40), supplemented with protease inhibitor cocktail (Roche). Protein concentration was determined using the Bradford method (Bio-Rad, Hercules, CA). Cell lysates (30 µg) were mixed with sample loading buffer (125 mM Tris-HCl [pH, 6.8], 4% [wt/vol] sodium dodecyl sulfate, 20% [vol/vol] glycerol, 200 mM dithiothreitol, 0.04% [wt/vol] bromphenol blue). Proteins were resolved on a sodium dodecyl sulfate-12% polyacrylamide gel and transferred to a Hybond-ECL nitrocel-lulose membrane (GE Healthcare, Freiburg, Germany). After electroblotting, the gel was stained with Ponceau S to verify uniform loading and transfer. Membranes were blocked with 5% (wt/vol) nonfat dry milk in PBS-T (PBS, 0.1% [vol/vol] Tween 20) for 2 hours at room temperature and subsequently incubated with desired primary antibody (diluted in PBS containing 5% nonfat milk and 0.1% Tween 20) overnight at 4°C with gentle agitation. After being washed three times with PBS containing 0.1% Tween 20, the membrane was incubated with horseradish peroxidase–coupled secondary antibody for 1 hour at room temperature. Chemiluminescence detection was performed by incubating the membranes with appropriate SuperSignal substrate (Pierce, Rockford, IL) followed by analyzing on a CCD cooling camera (Fuji LAS-1000plus, Fujifilm, Toyko).

The following antibodies were purchased: mouse monoclonal anti–proliferating cell nuclear antigen (PCNA) antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), mouse monoclonal anti-Bcl-2 antibody (Santa Cruz), mouse monoclonal anti-actin antibody (C4 [ICN Biomedicals, Irvine, CA]), and horseradish peroxidase–conjugated secondary antibodies (Pierce). The chemiluminescence was quantified with the aid of the Aida/two-dimensional densitometry program (Raytest Isotopenmeb, Straubenhardt, Germany).

Statistics
All statistical analyses were performed using the SigmaStat software package (Jandel Corp., San Rafael, CA). Results are expressed as mean ± standard deviation. To determine whether the various cryoprotective agents influenced cell survival after two different time points (24 hours and 1 week), two-way analysis of variance (ANOVA) was used. In case of statistically significant differences, Tukey’s test was used to determine which groups statistically differed from each other. Statistical significance was accepted if p < .05.

	RESULTS

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Viability and Apoptosis of Thawed NPCs
For measuring viability and early stages of apoptosis, neural precursors were maintained growing adherently. Comparing with neurospheres, this way of cultivation allowed more accurate counting and fluorometry of the cells. Necrotic cell death caused by freezing process was measured by the uptake of trypan blue, immediately after thawing and washing out cryoprotective agents. Cells with compromised plasma membranes permitted entry of the dye, whereas viable cells excluded the dye. After counting, we observed a maximum of 60% recovered cortical NPCs. In some cases, necrotic cell death was below 10% (10% DMSO + 0.2 M trehalose), but in most other cases, it did not exceed 30% (Fig. 1A). Samples frozen for 5 days in –70°C and in liquid nitrogen did not show any difference in viability (data not shown). The results were comparable for up to 1 month at –70°C and for up to 1 year in liquid nitrogen.

View larger version (22K): [in this window] [in a new window]   Figure 1. (A): Before freezing, neural precursor cells (NPCs) were expanded as monolayer (see Materials and Methods). Analysis of viability applying trypan blue dye exclusion test after freezing in a –70°C freezer is shown. Percentage of necrotic cell death ± standard error of the mean immediately after thawing is presented in the histogram. (B): Analysis of apoptosis by annexin-V. Fresh and thawed (taken from –70°C) cortical NPCs were stained with annexin-V fluorescein isothiocyanate and subjected to assessment by flow cytometry. The numbers (mean ± standard deviation) shown in the histogram represent the percentage of cells with exposed phosphatidylserine, indicative of early apoptosis. Abbreviations: DMSO, dimethyl sulfoxide; FCS, fetal calf serum.

Effect of Different Cryoprotectants on Neurosphere Survival Rate
Figure 2 displays the effects of various cryoprotective agents on cell survival analyzed via PI fluorescence-activated cell sorter after lysing the cells using the Nicoletti method [18]. We used 5% and 10% DMSO, 10% FCS, 10% glycerol, 0.2 M trehalose, or various combinations thereof. Statistical analysis revealed a significant effect of time (p < .001) and cryoprotectant (p < .001), as well as a significant interaction between time and cryoprotectant (p < .001). Subsequent post-hoc test showed that survival of unfrozen cells grown as free-floating neurospheres was approximately 70% and already within a 24-hour post-thawing interval significantly decreased in some indicated cases when multiple comparisons versus unfrozen cells were calculated (Jandel Sigma Stat 2.0, Dunnett’s method) (Fig. 2, asterisks). During the first week after thawing, cell survival was significantly reduced in all cases when compared with unfrozen control samples. However, the best results were obtained when 10% glycerol or 10% DMSO with or without trehalose was used as cryoprotectant (Fig. 2). Some cryoprotective agents conferred a good protection in a short-term setting (24 hours) but proved to be among the worst in a longer time setting (e.g., ethylene glycol).

View larger version (36K): [in this window] [in a new window]   Figure 2. Neural precursor cells were generated from mouse fetal forebrain, cultivated for 2 weeks in the presence of fibroblast growth factor-2 and epidermal growth factor, and subjected to freezing at –70°C in a rate-controlled manner. The cell survival was calculated after measuring the rate of apoptosis (sub-G1 population), 24 hours or 1 week after thawing of cultures using flow cytometry and cell-cycle analysis. Before cryopreservation, some neurospheres were treated with Accutase (Acc) for 15 minutes at 37°C to obtain single-cell populations. *p < .05 versus fresh (two-way analysis of variance). Abbreviations: DMSO, dimethyl sulfoxide; FCS, fetal calf serum.

Effects of Cryopreservation on Colony Formation
NPC recovery was assessed by traditional assay of colony formation. Viable single cells were grown, generating colonies. For each freeze/thaw sample, a value for total CFUs generated within 2 weeks after thawing was calculated as average from three independent experiments. Recovery was obtained as a ratio between CFU after freezing and CFU unfrozen control sample. The results were expressed as percent control and are presented in Figure 3. Recovery was approximately 26% and did not significantly differ between DMSO and glycerol frozen samples.

View larger version (18K): [in this window] [in a new window]   Figure 3. Recovery of frozen-thawed neural precursor cells explored via colony formation assay. Neurospheres were enzymatically treated with Accutase to obtain single-cell population and subjected to gradual freezing up to –70°C. After thawing, the cells were seeded in six-well plates to determine clonogenicity of frozen cells. Clonogenic survival was calculated in both fresh (control) and frozen samples and presented as percent control. The data show results with neurospheres counted in triplicate cultures. Abbreviation: DMSO, dimethyl sulfoxide.

View larger version (23K): [in this window] [in a new window]   Figure 4. Effects of the caspase inhibitor zVAD-fmk on the recovery of frozen-thawed murine neural precursor cells protected during freezing with 10% glycerol. zVAD-fmk was supplemented in indicated concentrations to the expansion medium (see Materials and Methods) for 2 days. The cell death was measured by fluorescence-activated cell sorter and calculated for survival. The cells with cas-pase inhibitor survived significantly better compared with untreated ones (*p < .05, one-way analysis of variance).

View larger version (30K): [in this window] [in a new window]   Figure 5. Proliferation and survival of fresh and frozen mouse neural precursor cell cultures. Frozen-thawed cells had been cultured for 2 weeks before protein extracts were taken from indicated samples. Proteins were subjected to PAGE and probed with antibodies directed to PCNA, Bcl-2, and actin as a loading control. The values obtained with densitometry, the expression of the proliferation (PCNA) and prosurvival (Bcl-2) markers, are presented in the histogram. Abbreviations: Acc, Accutase; DMSO, dimethyl sulfoxide; FCS, fetal calf serum; PCNA, proliferating cell nuclear antigen.

View larger version (59K): [in this window] [in a new window]   Figure 6. (A): Phase-contrast photomicrographs indicating fresh and frozen-thawed (10% glycerol) unfixed neurospheres. (B): Soon after withdrawal of mitogens (epidermal growth factor and fibroblast growth factor-2), frozen-thawed neurospheres, grown for 1 week, readily differentiated into major subtypes of the brain cells. One week after onset of differentiation, the cells were fixed and processed for immunocytochemical staining. Several phenotypes were identified using a combination of markers. 4'-6'-Diamidino-2-phenylindole (DAPI) stain (blue) was used to visualize nuclei, ßIII tubulin was an early marker expressed by immature neurons, the presence of glial fibrillary acid protein (GFAP) denoted glia, and a subset of oligodendrocytes expressed oligodendrocyte marker O4.

	DISCUSSION

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Cryopreservation of NPCs has not been studied in detail, except as a part of transplantation studies [6, 21–23]. The present study demonstrates that deep-freezing (–70°C) may allow banking of murine neurospheres, with preserved functionality yielding a minimum of 50% NPC recovery. In comparison, cryopreserva-tion of HSCs, which have been widely used clinically, retrieved 50%–60% HSCs [24, 25]. We used a rate-controlled freezing device, which was affordable and easy to handle. Murine NPCs readily proliferated in response to EGF and fibroblast growth factor-2, forming clonal structures described as neurospheres. The cells were gradually frozen and stored for 5 days at –70°C. For comparison, parallel samples were also stored in liquid nitrogen but did not reveal any significant difference with respect to viability [26]. We tested a broad spectrum of cryoprotective agents that have been successfully used to protect various mammalian cells, including primary cells, from freezing-related insults. Comparing the cell survival after freezing and thawing of neurospheres with fresh tissue, we noticed that the cell survival significantly decreased 24 hours after thawing. Because unfrozen neuro-spheres undergo spontaneous apoptosis during expansion [27], 1 week after thawing of murine neurospheres, cell survival is usually below 50%. We chose time points of 0 hours, 24 hours, and 1 week for assessment of cell survival. Immediately after thawing, there were necrotic cells due to cryopreservation stress, but surviving cells recovered well during the first 24 hours. Cell survival decreased during the first week after thawing. We did not include later time points because cell death or survival might then not be a consequence of the freezing process but of other factors (e.g., the size of the neurospheres). Both 5% DMSO and 10% DMSO were effective in preserving survival after 1 week. However, DMSO is known to be toxic, with side-effects after prolonged exposure [28]. In an attempt to find a less-toxic compound able to replace DMSO and exerting less toxicity, other protective agents were investigated. Although trehalose provided good protection when loaded into cells [15] or applied extracellularly [29], this sugar exhibited poor protection toward NPCs in our experiments. A somewhat superior effect was achieved in combination with DMSO, yielding least damage during freezing and within the first 24 hours of recovery. Although longer preincubation of the slowly penetrating agent glycerol has not been tested, it conferred a very good protection of murine NPCs with a short preincubation. Moreover, clonogenicity of frozen glycerol samples proved to be as good as clonogenicity of DMSO samples.

As an excellent marker for proliferation, we analyzed PCNA expression by Western blotting. Both 10% glycerol and 10% DMSO kept the proliferation levels on that one characteristic for freshly isolated cells. In contrast, the antiapoptotic protein Bcl-2 decreased using different freezing techniques but still remained on a relatively high level in samples frozen with DMSO or glycerol. Whether the observed Bcl-2 decline was a result of activation of caspases during cryopreservation is not clear from our experiments, but our previous investigations showed the decrease of Bcl-2 induced by DNA damage is secondary to the activation of apoptotic effector caspases [30]. Apoptosis is relevant to cryo-injury in several mammalian cell types [31, 32]. Our investigation confirmed previous observations that caspase inhibitors can help to prevent apoptosis associated with cryopreservation [9]. However, in our cell system, the caspase inhibitor zVAD-fmk is only helpful when coapplied with DMSO but not with trehalose. Broad-spectrum caspase inhibitor conferred better protection observed within the first week of revitalization but was ineffective in protecting the cells during freezing, suggesting prevalence of necrosis during the freezing process. Detailed investigations on the differentiation capacity of NPCs after freezing and thawing clearly indicate that NPCs were readily differentiated into both neurons and glia with similar relative amounts of glial and neuronal cells compared with fresh NPCs.

	CONCLUSION

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We conclude, first, that freezing at –70°C of murine forebrain NPCs results in a minimum of 50% cell survival within neurospheres after cryopreservation in either 10% DMSO or 10% glycerol and a minimum of 25% of clonogenic survival after cryopreservation in either 10% DMSO or 10% glycerol. Second, cell viability is required to exceed 70% before freezing for achieving approximately 50% cell survival after thawing. Third, caspase inhibitors increase viability during the post-thawing recovery (10–20 µM). Fourth, frozen-thawed NPCs retained their proliferative capacity and expressed specific markers of proliferation such as PCNA. Fifth, the antiapoptotic protein Bcl-2 decreased during cryopreservation. Sixth, the percentage of differentiated neurons did not change in post-thawed differentiated samples compared with fresh tissue. Finally, freezing the cells in liquid nitrogen did not change viability of examined cells compared with –70°C for up to 1 month. Taken together, our results argue that freezing of murine NPCs preserves cell properties and multilineage potential of NPCs and allows preparation of tissues for restorative therapy, granting necessary safety and quality control standards.

	ACKNOWLEDGMENTS

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We would like to thank Ute Roemuss and Annett Brandt for the preparation of NPCs and excellent technical assistance. Dr. Sigrid Schwarz is acknowledged for helpful discussions.

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Top Abstract Introduction Materials and Methods Results Discussion Conclusion References

 

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