Brought to you by Portland Press Ltd.
Published on behalf of the International Federation for Cell Biology
Cancer Cell death Cell cycle Cytoskeleton Exo/endocytosis Differentiation Division Organelles Signalling Stem cells Trafficking
Cell Biology International (2008) 32, 950–958 (Printed in Great Britain)
Proliferation, multipotency and neuronal differentiation of cryopreserved neural progenitor cells derived from the olfactory neuroepithelium of the adult rat
Tao Xuea, Li Qiaoa, Jianhua Qiua*, Lianjun Lua, Dingjun Zhaa and Li Weib
aDepartment of Otolaryngology, Xijing Hospital, the Fourth Military Medical University, Xi‘an 710032, PR China
bDepartment of Obstetrics and Gynecology, Xijing Hospital, the Fourth Military Medical University, Xi‘an 710032, PR China


Abstract

The use of olfactory neuroepithelium neural progenitor cells for transplantation has attracted attention in the treatment of many neurological disorders, which require efficient recovery methods and cryopreservation procedures. The purpose of this study was to evaluate different cryopreservation techniques for neural progenitor cells derived from the olfactory neuroepithelium (ONe NPCs) in adult rats. Initially, we compared the survival rates of cryopreserved ONe NPCs treated with six different cryoprotectants: dimethylsulfoxide (DMSO), ethylene glycol (EG) and glycerol, each with or without 10% FBS and with two different storage periods in liquid nitrogen (−196°C), specifically 3days short-term storage and 3months long-term storage. We assessed the recovery efficiency of ONe NPCs after freezing and thawing by viability testing and colony-forming assay as well as immunocytochemistry under different conditions. No significant difference in the survival rate was observed among these different cryoprotectants. With these protocols, ONe NPCs retained their multipotency and differentiated into glial (GFAP-positive), neuronal (NeuN-positive) and oligodendroglia (Galc-positive) cells. Collectively, our results imply that, under optimal conditions, ONe NPCs might be cryopreserved for periods of >3months without losing their proliferative and multipotency activities.


Keywords: Neural precursor cell, Cryopreservation, Olfactory neuroepithelium.

*Corresponding author. Tel.: +86 29 84775381; fax: +86 29 83224744.


1 Introduction

Stem cells are undifferentiated cells that have the ability to undergo numerous divisions and self-renewal in culture; in addition, they are able to differentiate into multilineage, functionally specialized cells. The replacement of lost or damaged neurons by neural stem cells (NSCs) or neural progenitor cells(NPCs) represents great promise for clinical treatment of many neurological disorders (Chiasson et al., 1999; Loeffler and Roeder, 2002; Weiss et al., 1996). NSCs, with their capacity for unlimited self renewal and production of non-restricted lineage- committed progenitors in contrast to neural ‘progenitor’ or ‘precursor’ cells, have long been thought of as central to the repair and regeneration processes of replacing cells lost in many devastating diseases. Multipotent NSCs can be found in the embryonic, neonatal and adult mammalian CNS (Reynolds and Weiss, 1992). The forebrain subventricular zone (SVZ) and dentate gyrus are considered to be the major sources of self-renewing, multipotent NSCs. Furthermore, multipotential precursors with stem cell features can be isolated not only from the SVZ, but also from the entire rostral extension, including the distal portion within the olfactory bulb (OB) (Gritti et al., 2002; Otaegi et al., 2007).

Recently, the regenerative capacity of the olfactory system has attracted attention. Olfactory ensheathing cells (OECs) from adult OBs have been used to provide limited axonal regeneration (Dombrowski et al., 2006; Lu et al., 2006; Ramer et al., 2004) and to repair demyelinated regions of the CNS (Lakatos et al., 2000; Nieto-Sampedro, 2003; Santos-Benito and Ramon-Cueto, 2003). However, the harvest of OECs from the olfactory bulb involves highly invasive surgery, making this a problematic source for clinical therapy (Barnett et al., 2000; Deng et al., 2006; Lopez-Vales et al., 2007; Lu et al., 2002). The olfactory neuroepithelium (ONe) undergoes lifelong repair by progenitors, which are capable of replacing both neuronal and supporting cells (Beites et al., 2005; Calof and Chikaraishi, 1989; Hahn et al., 2005). Previously, some authors (Beites et al., 2005; Chuah et al., 1991) developed procedures for isolation and culture of ONe-derived neurosphere-forming cells from adult rats that remain mitotically active and have the characteristics of neural progenitor cells (NPCs). From a developmental standpoint, murine ONe NPCs represent an accessible and important system for the study of basic ONe NPC properties such as self-renewal and multipotency. The clinical implications of ONe NPCs are potentially profound. Cryopreservation is 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 NSCs has been done using dimethyl sulfoxide (DMSO). However, the role of different cryoprotectants in the preservation of ONe NPCs has not been studied in detail. Successful long-term storage and preservation of ONe NPCs is an important prerequisite for their potential therapeutic application in regenerative approaches such as transplantation.

We believe that ONe NPCs may be an ideal source for autologous NSC transplantation and will eventually be used clinically. The widespread use of various treatment protocols using ONe NPCs have resulted in an increased demand for cryobiological techniques for storing these cells. The need has led to a re-examination of many cryobiological practices. However, DMSO is known to be toxic, with side-effects after prolonged exposure (Kim et al., 2007; Syme et al., 2004). Therefore, developing a cryopreservation medium with little toxicity is mandatory. In this paper we show that ONe NPC cultures derived from the adult rat can be cryopreserved. This enables the investigator to produce large numbers of NPCs from a small starting population of ONe NPCs and freeze these NPCs for later use. The ability to cryopreserve ONe NPCs greatly simplifies the use of this cell culture system.

2 Materials and methods

2.1 Animals and reagents

Male and female Sprague–Dawley rats, 200–250g, were used. Animals were housed in a temperature and humidity controlled room that was maintained on a 12-h light/dark cycle and had free access to food and water. All procedures were approved by the Animal Welfare Committee of the Fourth Military Medical University. All efforts were made to minimize the number of animals used and their suffering.

Fibroblast growth factor-2 (FGF-2) and epidermal growth factor (EGF) were purchased from PeproTech (USA) and B27 was purchased from Invitrogen (CA, USA). The CCK-8 kit was purchased from Dojindo Laboratories (Japan). Others reagents for tissue culture were purchased from HyClone (USA) and Sigma (USA).

2.2 Removal of the olfactory neuroepithelium tissue

On the first day of the experiment, rats were anesthetized by an i.p. injection of chloral hydrate 0.3M (0.8ml/100g). Ten milliliters of an aqueous solution of 2% ZnSO4 were applied into the two nares of each animal (Michel et al., 1999). Twelve hours later, rats were deeply anesthetized with sodium pentobarbital. A midline skin incision was made in the nasal bone, and a burr hole was drilled. A piece of tissue including the olfactory neuroepithelium (Fig. 1A, white arrowheads) was transferred into phosphate-buffered saline (PBS) and cut into small pieces under a surgical microscope. A piece of bone wax was used to seal the nasal bone defect and the skin was closed with coated vicryl. The ONe NPCs of the adult rats were prepared as described previously (Chuah et al., 1991; Hahn et al., 2005). Briefly, cells from adult rats were incubated with 1.4μg/μl trypsin, 0.7μg/μl hyaluronidase and 0.2μg/μl kynurenic acid for 15min at 37°C and triturated using a fire-polished Pasteur pipette, the cells were seeded in the culture medium as described below.


Fig. 1

A midline skin incision was made in the nasal bone and a burr hole was drilled. A piece of tissue including the olfactory neuroepithelium (white arrowheads) was transferred into phosphate-buffered saline (PBS) and cut into small pieces under a surgical microscope (A). Three days after plating, some rounded cells started to divide. Scale bar corresponds to 100μm (B). A smaller proportion of the cells differentiated into neuron-like cells. Scale bar corresponds to 50μm (C). Daily inspection of the cultures showed that the rounded cells divided, forming cell aggregates or spheres, which reached confluence in the presence of the mitogens by the 7th day. Scale bar corresponds to 100μm (D).


2.3 Olfactory neuroepithelium NPC culture medium

ONe NPCs were seeded in Dulbecco's modified Eagle's medium/F12 (DMEM/F12) with heat-inactivated 10% fetal bovine serum (FBS) (Invitrogen Cat No. 10099-141) containing 0.6% glucose, 1:50 B27, 2nM NaHCO3, 0.5mM HEPES, 100μg/ml human apo-transferrin, 60μM putrescine, 20nM progesterone, 30nM selenium chloride, 25μg/ml human insulin, 2μM l-glutamine, 20ng/ml FGF-2 and 20ng/ml EGF. Cells were grown in uncoated plastic flasks during the primary culture as free-floating clusters (neurospheres). Neural stem/progenitor cell populations were obtained from the neurospheres after at least two passages at 37°C in a humidified 5% CO2 in air atmosphere.

To initiate differentiation, after FGF-2 and EGF were removed, colonies of ONe NPCs were plated for 10days on glass coverslips precoated with 0.1mg/ml poly-l-lysine in DMEM/F12/B27 containing 10% FBS at a density of 100,000cells/cm2. The cells were then fixed with 4% paraformaldehyde to assess whether these dividing cells could differentiate into neurons, astrocytes and oligodendrocytes.

2.4 Cryopreservation techniques and post-thawed

For the cryopreservation of ONe NPCs populations, cells were prepared as described above. After at least two passages of neurospheres, the samples were resuspended in special cryoprotectants, stored in a freezer at −70°C, and 24h later the parallel samples were transferred to liquid nitrogen (−196°C). Here, 6 different cryoprotectants were applied, which were mainly made up with serum-free expansion medium. Moreover, each was supplemented with different cryoprotective additives (DMSO, glycerol, ethylene glycol), as well as with or without 10% FBS. After the 3-day or 3-month freezing period, the cells were taken from the liquid nitrogen, thawed rapidly in a 37°C water bath with continuous agitation and diluted with the culture medium to 10 times the volume. Four hours later, the freezing medium was replaced with the fresh serum-free expansion medium and neurospheres were maintained as described above.

2.5 Measurement of cell viability and cell proliferation assay

To measure trypan blue exclusion as described by Milosevic et al. (2005), fresh or frozen-thawed ONe NPCs were incubated in triplicates in a 0.25% dye solution (Gibco). Total cell numbers and the number of trypan blue positive cells was then determined by counting at least 200 cells per sample in a hemocytometer. Cell viability (percentage) is the ratio of the number of trypan blue-impermeable cells to the total cell count (trypan blue-impermeable cell number/total cell number). Cells were observed under a microscope and counted as stained and nonstained cells on hemocytometer separately, then the viable cell ratios were calculated according to the following formula: viable cell ratio (%)=(nonstained cells number/total cells number)×100%. Necrotic cells (percentage) refer to the percentage of trypan blue-positive cells.

To measure CCK-8, fresh or frozen-thawed ONe NPCs in the above, different cryoprotective additives were monitored via an inverted microscope and their viability was detected every 48h using a CCK-8 kit, which calculates cell survival according to the protocols of CCK-8, where ODexpt is the optical density value of ONe NPCs in the above different cryoprotective additives.

2.6 Clonogenic survival assay

ONe NPC survival after exposure to the freezing process with different cryoprotectants was measured via the colony-forming assay as described by Davis and Temple (1994). Clonogenicity was determined by measuring colony formation as described by Lu and Wong (2005) and Zappone et al. (2000). In brief, after the cells were thawed and washed, ONe NPCs were adjusted to a final concentration of 5×104/ ml in a 9ml culture medium. The cells were seeded in triplicate into 6-well plates. Relative survival value was calculated by assessing the ratio of colony-forming units (CFUs) of secondary neurospheres, which were treated with different cryopreservation techniques and thawed as mentioned before, then exposed to the fresh culture samples for 14days at 37°C in a humidified 5% CO2 in air atmosphere. Secondary neurospheres were scored using an inverted microscope applying standard criteria for their identification.

2.7 BrdU labeling and neurosphere differentiation

To label the cells as described by Namba et al. (2007) and Lei et al. (2007), 6μg/ml 5-bromodeoxyuridine (BrdU, sigma) was added to the medium to label dividing neurospheres for 24h. The BrdU-labeled neurospheres were trypsinized and cultured on a poly-l-lysine-coated 24-well plate, with DMEM/F12/ B27 containing 10% FBS, without FGF-2 and EGF. After 5days, the mixed cultures were fixed for an immunofluorescent double-labeling assay as described below.

2.8 Immunocytochemistry and immunofluorescence

The expression of each antigen was examined in separate experiments at least three times. After each treatment, cells were fixed with 4% paraformaldehyde for 15min at room temperature and washed three times with 0.01M PBS. They were permeabilized in 1% Triton X-100 for 15min and rinsed three times with 0.01M PBS. Non-specific binding was blocked by a 1-h treatment in 5% normal goat serum. The cells were incubated with primary antibodies at 4°C overnight at room temperature. Sources and dilution of primary antibodies were as follows: mouse anti-nestin (1:200; Chemicon, USA; monoclonal antibody to label neuroepithelial stem cells), mouse anti-neuron specific nuclear protein (anti-NeuN, 1:200; Chemicon, USA; monoclonal antibody to label neurons), mouse anti-glial fibrillate acid protein (anti-GFAP, 1:100; Chemicon, USA; monoclonal antibody to label astrocytes), mouse anti-calactocerebroside (Anti-GalC, 1:100; Chemicon, USA; monoclonal antibody to label oligodendrocytes). After three washes in 0.01M PBS, cells were incubated with biotinylated goat anti-mouse or goat anti-rabbit IgG (1:100; Boster Biological Technology Co, Wuhan, China;) for 2h at room temperature, followed by 2h of incubation in avidin–biotinylated peroxidase complex (1:100; Boster, Wuhan, China) at room temperature. Diaminobenzidine (DAB; Sigma; 0.05%) was used as a chromogen, with reactions sustained for 5–15min at room temperature and in the dark. For negative controls, replacing the primary antibodies with normal goat serum or staining without secondary antibody was investigated in each experiment, and in either case no specific positive staining was detected.

For the BrdU/NeuN or BrdU/GFAP immunofluorescent double-labeling, the fixed cells were incubated in 2N HCl at 37°C for 30min, followed by incubation in borate buffer (pH 8.4) for 20min. The cells were subsequently incubated overnight at 4°C with the following primary antibodies: rat anti-BrdU (1:3000; Chemicon, USA), mouse anti-NeuN, and mouse anti-GFAP, as mentioned above. After rinsing with 0.01M PBS three times, the cells were incubated with the following secondary antibodies: FITC-conjugated goat anti-rabbit IgG (1:50; Boster, Wuhan, China) and Cy3-conjugated goat anti-mouse IgG (1:50; Boster, Wuhan, China) or Rhodamine-conjugated goat anti-mouse IgG (1:50; Zhongshan Biotechnology Co, Beijing, China) for 1h at room temperature. Labeled cells were observed on an Olympus optic microscope (Tokyo, Japan), using appropriate fluorescence filters, and were imaged using a SPOT II camera (DP70, Olympus, Tokyo, Japan).

2.9 Statistics

All statistical analyses were performed using the SPSS 11.0 (Jandel Corp, San Rafael, CA, USA). Results are expressed as mean±SD. In case of statistically significant differences, a Tukey's test was used to determine which groups statistically differed from each other. One-way analysis of variance (ANOVA) procedures were employed to assess the effect of average percentage differences in cell recovery in different experimental methodologies. Statistical significance was accepted if P<0.05.

3 Results

3.1 Isolation and characterization of ONe NPCs

To isolate ONe NPCs, a cell suspension was prepared from adult rat olfactory neuroepithelium tissue and plated under tissue culture conditions in the presence of FGF-2 and EGF. Three days after plating, some rounded cells started to divide (Fig. 1B, black arrowheads). A smaller proportion of the cells differentiated into neuron- like cells (Fig. 1C, black arrowheads). Daily inspection of the cultures showed that the rounded cells divided, forming cell aggregates or spheres (Moses et al., 2006; Reynolds and Weiss, 1996), reaching confluence in the presence of the mitogens by the 7th day (Fig. 1D).

Under these standard growth conditions, cells could be passaged every 7–9days for at least 8months for a total of 30 passages. The increase in cell numbers varied between passages, but a tendency toward lower proliferation was observed in the later passages. Immunocytochemistry analyses revealed that ONe NPCs expressed nestin (Fig. 2A), a marker of neural stem cells. To test whether these proliferative precursor cells possessed stem cell features such as multipotentiality and self-renewal capacity, 12 clonally-derived secondary spheres and 12 tertiary spheres were analyzed by triple immunostaining. The spheres generated neurons, astrocytes and oligodendrocytes after differentiation, indicating that the ONe NPCs were multipotent (Fig. 2B–D). These results indicate that ONe NPCs were predominantly composed of highly proliferative neural precursor cells, which could be expanded for long periods of time and were multipotent with self-renewal capacity.


Fig. 2

Immunocytochemistry analyses revealed that ONe NPCs formed neurospheres. ONe NPCs formed neurospheres expressed nestin. Scale bar corresponds to 50μm (A). Soon after withdrawal of mitogens (EGF and FGF-2), ONe NPCs neurospheres, grown for 10days, readily differentiated into major subtypes of the brain cells. Ten days after onset of differentiation, the cells were fixed and processed for immunocytochemical staining. NeuN was an marker expressed by mature neurons (B); the presence of glial fibrillary acid protein (GFAP) denoted glia (C); and a subset of oligodendrocytes expressed oligodendrocyte marker GalC (D). Scale bar corresponds to 100μm.


3.2 Viability of cryopreserved ONe NPCs

Necrotic cell death caused by the cryopreservation 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 necrotic cell death was below 30% in most cases (Fig. 3A). Samples frozen for 3days and 3months did not show any difference in viability (Fig. 3B). There was no significant difference of ONe NPC cell survival among the six cryopreserved groups and the fresh ONe NPC group (the negative control). The growth curve of ONe NPCs with CCK-8 assays is shown in Fig. 3C. Similarly, there was no significant difference in the ONe NPC growth curve among the seven groups.


Fig. 3

Before freezing, ONe NPCs were expanded as a monolayer (see Section 2). Analysis of viability applying trypan blue dye exclusion test after freezing is shown. Percentage of necrotic cell death±standard error of the mean immediately 3days after thawing is presented in the histogram (A). Before freezing, ONe NPCs were expanded as monolayer (see Section 2). Analysis of viability applying trypan blue dye exclusion test after freezing is shown. Percentage of necrotic cell death±standard error of the mean immediately 3months after thawing is presented in the histogram (B). The cell viability of ONe NPCs was analyzed using CCK-8 assay. There was no significant difference of the ONe NPCs' growth curve between the seven groups. Growth curve of ONe NPCs cells: 104 NSCs were plated on 6cm culture dishes in DMEM/F12/B27 without FGF-2 and EGF. After culturing for 14days, the absorbance was measured at 450nm using a microplate reader. The growth curve did not significantly differ between the different cryoprotective additives (C). Recovery of frozen-thawed ONe NPCs explored via colony formation assay. 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 a percentage control. The data show results with neurospheres counted in triplicate cultures (D).


Cryopreserved and thawed ONe 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 2weeks was calculated as an average from three independent experiments. Recovery rate was obtained as a ratio between the CFU of cryopreserved ONe NPCs and the CFU of the unfrozen control sample. The results were expressed as percentages(Fig. 3D). The recovery rate of the seven groups was &007E;26%, and showed no obvious difference from each other.

3.3 Differentiation of cryopreserved treated ONe NPCs

Within the first week of the post-thawing period, ONe NPCs formed neurospheres identical to those seen in the fresh tissue (Fig. 4G). After removal of growth factors, cryopreserved and thawed neurospheres readily differentiated into neurons (NeuN-positive), astrocytes (GFAP-positive), as detected by Immunofluorescent double labeling (Fig. 4A–F). The relative number of neurons was estimated by calculating the percentage of NeuN-positive vs. BrdU-labeled cells. On the one hand, the number of viable cells was markedly reduced after the freeze–thaw process. However, on the other hand, the relative number of neurons in the cryopreserved and thawed tissue showed no statistical difference from the fresh untreated one: 8.26±0.96 in 10% DMSO; 7.21±0.32 in 10% DMSO+10% FBS; 7.56±0.27 in 10% glycerol; 7.17±0.81% in 10% glycerol+10% FBS; 6.97±0.67% in 10% ethylene glycol; 7.31±1.02% in 10% ethylene glycol+10% FBS; and 7.26±1.2% in the fresh sample (Fig. 4H).


Fig. 4

Neural differentiation of ONe NPCs after thawing. Frozen-thawed ONe NPCs were labeled by BrdU for 3days and cultured for 5days. At 5days, a few BrdU-positive ONe NPCs expressed NeuN (arrows). BrdU, green; NeuN, red. Scale bar corresponds to 50μ M (A–C). At 5days, some BrdU-positive ONe NPCs expressed GFAP (arrows). BrdU, green; GFAP, red. Scale bar corresponds to 50μM (D–F). Phase-contrast photomicrographs indicating frozen-thawed (10% glycerol) unfixed neurospheres. Scale bar corresponds to 100μM (G). The relative number of neurons in the cryopreserved and thawed tissue was not statistically different from fresh tissue and ranged as follows: 8.26±0.96 in 10% DMSO; 7.21±0.32 in 10% DMSO+10% FBS; 7.56±0.27 in 10% glycerol; 7.17±0.81% in 10% glycerol+10% FBS; 6.97+0.67% in 10% ethylene glycol; 7.31+1.02% in 10% ethylene glycol+10% FBS; and 7.26±1.2% in the fresh sample (H).


4 Discussion

Successful long-term storage and preservation of stem cells is an important precondition for their potential therapeutic application in regenerative approaches such as transplantation. The use of cryopreserved NSCs/NPCs in gene and cell therapy also requires preservation of their differentiation and proliferative capacities. In this light, it is necessary to assess whether the process of cryopreservation affects either the proliferative capacity of NSCs/NPCs or their developmental potential. The aim of various cryopreservation procedures is to minimize cell injury during the freeze–thaw process. Cell injury may result from extensive cellular dehydration (‘solution effect’) and/or intracellular ice crystallization (‘mechanical cell damage’) (Mazur, 1966; Meryman, 1956; Zhao et al., 2001). Today, the cryopreservation of adult human bone marrow mesenchymal stem cells (hMSC) is considered to offer not only a population of pluripotential cells but also an accessible means for establishing an abundant hMSC reservoir for further experimentation (Kotobuki et al., 2005; Xiang et al., 2007). Indeed, the different storage conditions used for NSCs/NPCs show a difference in cost and availability. Thus, the search for optimal freezing and storing procedures is a pivotal prerequisite for the general and systematic application of these cells in both preclinical and clinical settings. Similarly, with cryopreservation yields of 50–60% for HSCs, which are widely used clinically (Querol et al., 2000; Yang et al., 2003), our study demonstrates that deep-freezing at −196°C may allow banking of murine neurospheres, with a minimum of 50% of NPCs recovered with preserved functionality. ONe NPCs readily proliferated in response to EGF and FGF-2, forming clonal structures described as neurospheres. The choice of the cryoprotective agent and its optimal concentration is important for the required cell recovery after thawing (Lovelock and Bishop, 1959; McGann, 1978; Meryman, 1971). In this study, the cells were gradually frozen and stored at −70°C; subsequently, parallel samples were stored in liquid nitrogen for 3°days and 3°months. Then, we estimated the efficacy of different cryoprotective additives, as well as their roles in the recovery of the differentiative ability of cryopreserved ONe NPCs. Generally speaking, the most commonly used cryoprotectant, DMSO, may penetrate cells rapidly and avoid intracellular freezing by preventing a substantial increase in the external osmotic pressure, thus reducing ice crystal formation. However, DMSO is known to be toxic, with side-effects after prolonged exposure (Damon et al., 2006; Syme et al., 2004; Watanabe et al., 2006). Our data indicates that similar recovery results were obtained using five separate cryoprotective additives. Each cryoprotective additive – 10% DMSO or 10% glycerol and 10% ethylene glycol, with or without 10%FBS – maintained proliferation levels as well as the character of freshly isolated cells. Although evaluation of a longer preincubation of the slowly penetrating agent glycerol in liquid nitrogen has not been carried out, we have proved that it allows high-quality protection of ONe NPCs. Moreover, the clonogenicity of frozen glycerol samples proved to be as good as the clonogenicity of DMSO samples.

Frozen cells are injured by the direct effect of low temperature and the formation of ice crystals. On the one hand, intracellular ice formation coupled with a high rate of cooling may rupture the cell; on the other hand, extracellular ice formation results in increased extracellular medium osmolality as water is taken up, causing severe dehydration (Balint et al., 1999; Broxmeyer et al., 2003; Buchanan et al., 2004; Meyer et al., 2006). On the other hand, rate-controlled cryopreservation reduces this potential damage (Zhao et al., 2001). Although our results indicated that both techniques performed equally well, rate-controlled freezing should be explored in further studies.

If the freezing procedure is optimal and no cell damage has occurred, no further damage would be expected during the storage period (Katayama et al., 1997). 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 that there was no statistical difference between the mean cell recovery rate after cryopreservation in liquid nitrogen for 3days or 3months. In clonogenic assays, such as the BFU-E and CFU-GM assays, it is not possible to evaluate the total cell recovery rate due to the extreme variability of the colony appearance. Along this line, here we used clonogenic assays only to check the cell colony formation ability.

The critical issue of cryopreservation is whether frozen-thawed ONe NPCs are able to proliferate and to differentiate into neurons, astrocytes and oligodendrocytes (Hancock et al., 2000; Milosevic et al., 2005). Our objectives were to analyze the effect of cryopreservation on the survival of ONe NPCs and to establish whether cryopreserved ONe NPCs are able to proliferate in tissue culture while maintaining their proliferation and multipotency activity. The results obtained in this study indicate no statistically significant difference in ONe NPC multipotency activity between fresh cells and frozen-thawed cells. Furthermore, we obtained the same survival rate in the six methods studied. The neural phenotype of the cells which differentiated from cryopreserved ONe NPCs was estimated by immunofluorescence assay of neuron markers (NeuN) and astrocytes markers (GFAP). A previous study by See et al. (2004) showed that GalC was reported to rapidly disappear from the surface of cultured oligodendrocytes; our immunocytochemistry assay and other studies (Othman et al., 2005) have all proved that the ability of the cells to differentiate into oligodendrocytes was lower than their ability to differentiate into neurons or astrocytes, and only a minority of them were GalC positive. Thus, it was very difficult to get a positive result in the immunofluorescence assay and the cells were not estimated by the immunofluorescence assay of GlaC. Upon withdrawal of EGF and FGF-2, the thawed ONe NPCs cultures differentiated into neuronal cultures which showed the same overall appearance as the ones untreated by cryopreservation.

We have shown that ONe NPCs can be cryopreserved for long periods. Moreover, we have demonstrated that cryopreserved ONe NPCs can differentiate into neural cells which expressed neuron-specific gene products. In conclusion, our study describes excellent cryopreservation techniques for ONe NPCs which may not only preserve cell proliferation properties, but also maintain the multi-differentiative potential. It is tempting to speculate that we have found an excellent way for preparing tissue used for stem cell restorative therapy which meets the necessary safety and quality control standards.

Acknowledgements

This work was supported by grants to Prof. Jianhua Qiu from the National Natural Science Foundation of China (No. 30572023).

References

Balint B, Ivanovic, Z, Petakov, M, Taseski, J, Jovcic, G, Stojanovic, N. The cryopreservation protocol optimal for progenitor recovery is not optimal for preservation of marrow repopulating ability. Bone Marrow Transplant 1999:23:613-9
Crossref   Medline   1st Citation  

Barnett SC, Alexander, CL, Iwashita, Y, Gilson, JM, Crowther, J, Clark, L. Identification of a human olfactory ensheathing cell that can effect transplant-mediated remyelination of demyelinated CNS axons. Brain 2000:123:Pt 8:1581-8
Medline   1st Citation  

Beites CL, Kawauchi, S, Crocker, CE, Calof, AL. Identification and molecular regulation of neural stem cells in the olfactory epithelium. Exp Cell Res 2005:306:309-16
Crossref   Medline   1st Citation   2nd  

Broxmeyer HE, Srour, EF, Hangoc, G, Cooper, S, Anderson, SA, Bodine, DM. High-efficiency recovery of functional hematopoietic progenitor and stem cells from human cord blood cryopreserved for 15years. Proc Natl Acad Sci U S A 2003:100:645-50
Crossref   Medline   1st Citation  

Buchanan SS, Gross, SA, Acker, JP, Toner, M, Carpenter, JF, Pyatt, DW. Cryopreservation of stem cells using trehalose: evaluation of the method using a human hematopoietic cell line. Stem Cells Dev 2004:13:295-305
Crossref   Medline   1st Citation  

Calof AL, Chikaraishi, DM. Analysis of neurogenesis in a mammalian neuroepithelium: proliferation and differentiation of an olfactory neuron precursor in vitro. Neuron 1989:3:115-27
Crossref   Medline   1st Citation  

Chiasson BJ, Tropepe, V, Morshead, CM, van der Kooy, D. Adult mammalian forebrain ependymal and subependymal cells demonstrate proliferative potential, but only subependymal cells have neural stem cell characteristics. J Neurosci 1999:19:4462-71
Medline   1st Citation  

Chuah MI, David, S, Blaschuk, O. Differentiation and survival of rat olfactory epithelial neurons in dissociated cell culture. Brain Res Dev Brain Res 1991:60:23-32
1st Citation   2nd  

Damon L, Rugo, H, Tolaney, S, Navarro, W, Martin, T, Ries, C. Cytoreduction of lymphoid malignancies and mobilization of blood hematopoietic progenitor cells with high doses of cyclophosphamide and etoposide plus filgrastim. Biol Blood Marrow Transplant 2006:12:316-24
Crossref   Medline   1st Citation  

Davis AA, Temple, S. A self-renewing multipotential stem cell in embryonic rat cerebral cortex. Nature 1994:372:263-6
Crossref   Medline   1st Citation  

Deng C, Gorrie, C, Hayward, I, Elston, B, Venn, M, Mackay-Sim, A. Survival and migration of human and rat olfactory ensheathing cells in intact and injured spinal cord. J Neurosci Res 2006:83:1201-12
Crossref   Medline   1st Citation  

Dombrowski MA, Sasaki, M, Lankford, KL, Kocsis, JD, Radtke, C. Myelination and nodal formation of regenerated peripheral nerve fibers following transplantation of acutely prepared olfactory ensheathing cells. Brain Res 2006:1125:1-8
Crossref   Medline   1st Citation  

Gritti A, Bonfanti, L, Doetsch, F, Caille, I, Alvarez-Buylla, A, Lim, DA. Multipotent neural stem cells reside into the rostral extension and olfactory bulb of adult rodents. J Neurosci 2002:22:437-45
Medline   1st Citation  

Hahn CG, Han, LY, Rawson, NE, Mirza, N, Borgmann-Winter, K, Lenox, RH. In vivo and in vitro neurogenesis in human olfactory epithelium. J Comp Neurol 2005:483:154-63
Crossref   Medline   1st Citation   2nd  

Hancock CR, Wetherington, JP, Lambert, NA, Condie, BG. Neuronal differentiation of cryopreserved neural progenitor cells derived from mouse embryonic stem cells. Biochem Biophys Res Commun 2000:271:418-21
Crossref   Medline   1st Citation  

Katayama Y, Yano, T, Bessho, A, Deguchi, S, Sunami, K, Mahmut, N. The effects of a simplified method for cryopreservation and thawing procedures on peripheral blood stem cells. Bone Marrow Transplant 1997:19:283-7
Crossref   Medline   1st Citation  

Kim DH, Jamal, N, Saragosa, R, Loach, D, Wright, J, Gupta, V. Similar outcomes of cryopreserved allogeneic peripheral stem cell transplants (PBSCT) compared to fresh allografts. Biol Blood Marrow Transplant 2007:13:1233-43
Crossref   Medline   1st Citation  

Kotobuki N, Hirose, M, Machida, H, Katou, Y, Muraki, K, Takakura, Y. Viability and osteogenic potential of cryopreserved human bone marrow-derived mesenchymal cells. Tissue Eng 2005:11:663-73
Crossref   Medline   1st Citation  

Lakatos A, Franklin, RJ, Barnett, SC. Olfactory ensheathing cells and Schwann cells differ in their in vitro interactions with astrocytes. Glia 2000:32:214-25
Crossref   Medline   1st Citation  

Lei Z, Yongda, L, Jun, M, Yingyu, S, Shaoju, Z, Xinwen, Z. Culture and neural differentiation of rat bone marrow mesenchymal stem cells in vitro. Cell Biol Int 2007:31:916-23
Crossref   Medline   1st Citation  

Loeffler M, Roeder, I. Tissue stem cells: definition, plasticity, heterogeneity, self-organization and models – a conceptual approach. Cells Tissues Organs 2002:171:8-26
Crossref   Medline   1st Citation  

Lopez-Vales R, Fores, J, Navarro, X, Verdu, E. Chronic transplantation of olfactory ensheathing cells promotes partial recovery after complete spinal cord transection in the rat. Glia 2007:55:303-11
Crossref   Medline   1st Citation  

Lovelock JE, Bishop, MW. Prevention of freezing damage to living cells by dimethyl sulphoxide. Nature 1959:183:1394-5
Crossref   Medline   1st Citation  

Lu F, Wong, CS. A clonogenic survival assay of neural stem cells in rat spinal cord after exposure to ionizing radiation. Radiat Res 2005:163:63-71
Crossref   Medline   1st Citation  

Lu J, Feron, F, Mackay-Sim, A, Waite, PM. Olfactory ensheathing cells promote locomotor recovery after delayed transplantation into transected spinal cord. Brain 2002:125:14-21
Crossref   Medline   1st Citation  

Lu P, Yang, H, Culbertson, M, Graham, L, Roskams, AJ, Tuszynski, MH. Olfactory ensheathing cells do not exhibit unique migratory or axonal growth-promoting properties after spinal cord injury. J Neurosci 2006:26:11120-30
Crossref   Medline   1st Citation  

Mazur P. Theoretical and experimental effects of cooling and warming velocity on the survival of frozen and thawed cells. Cryobiology 1966:2:181-92
Crossref   Medline   1st Citation  

McGann LE. Differing actions of penetrating and nonpenetrating cryoprotective agents. Cryobiology 1978:15:382-90
Crossref   Medline   1st Citation  

Meryman HT. Cryoprotective agents. Cryobiology 1971:8:173-83
Crossref   Medline   1st Citation  

Meryman HT. Mechanics of freezing in living cells and tissues. Science 1956:124:515-21
Crossref   Medline   1st Citation  

Meyer TP, Hofmann, B, Zaisserer, J, Jacobs, VR, Fuchs, B, Rapp, S. Analysis and cryopreservation of hematopoietic stem and progenitor cells from umbilical cord blood. Cytotherapy 2006:8:265-76
Crossref   Medline   1st Citation  

Michel V, Monnier, Z, Cvetkovic, V, Math, F. Organotypic culture of neuroepithelium attached to olfactory bulb from adult mouse as a tool to study neuronal regeneration after ZnSO4 neuroepithelial trauma. Neurosci Lett 1999:271:195-8
Crossref   Medline   1st Citation  

Milosevic J, Storch, A, Schwarz, J. Cryopreservation does not affect proliferation and multipotency of murine neural precursor cells. Stem Cells 2005:23:681-8
Crossref   Medline   1st Citation   2nd  

Moses D, Teper, Y, Gantois, I, Finkelstein, DI, Horne, MK, Drago, J. Murine embryonic EGF-responsive ventral mesencephalic neurospheres display distinct regional specification and promote survival of dopaminergic neurons. Exp Neurol 2006:199:209-21
Crossref   Medline   1st Citation  

Namba T, Mochizuki, H, Onodera, M, Namiki, H, Seki, T. Postnatal neurogenesis in hippocampal slice cultures: early in vitro labeling of neural precursor cells leads to efficient neuronal production. J Neurosci Res 2007:85:1704-12
Crossref   Medline   1st Citation  

Nieto-Sampedro M. Central nervous system lesions that can and those that cannot be repaired with the help of olfactory bulb ensheathing cell transplants. Neurochem Res 2003:28:1659-76
Crossref   Medline   1st Citation  

Otaegi G, de Pablo, F, Vicario-Abejon, C, de la Rosa, EJ. Retinal and olfactory bulb precursor cells show distinct responses to FGF-2 and laminin. Cell Biol Int 2007:31:752-8
Crossref   Medline   1st Citation  

Othman M, Lu, C, Klueber, K, Winstead, W, Roisen, F. Clonal analysis of adult human olfactory neurosphere forming cells. Biotech Histochem 2005:80:189-200
Crossref   Medline   1st Citation  

Querol S, Capmany, G, Azqueta, C, Gabarro, M, Fornas, O, Martin-Henao, GA. Direct immunomagnetic method for CD34+ cell selection from cryopreserved cord blood grafts for ex vivo expansion protocols. Transfusion 2000:40:625-31
Crossref   Medline   1st Citation  

Ramer LM, Au, E, Richter, MW, Liu, J, Tetzlaff, W, Roskams, AJ. Peripheral olfactory ensheathing cells reduce scar and cavity formation and promote regeneration after spinal cord injury. J Comp Neurol 2004:473:1-15
Crossref   Medline   1st Citation  

Reynolds BA, Weiss, S. Clonal and population analyses demonstrate that an EGF-responsive mammalian embryonic CNS precursor is a stem cell. Dev Biol 1996:175:1-13
Crossref   Medline   1st Citation  

Reynolds BA, Weiss, S. Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system. Science 1992:255:1707-10
Crossref   Medline   1st Citation  

Santos-Benito FF, Ramon-Cueto, A. Olfactory ensheathing glia transplantation: a therapy to promote repair in the mammalian central nervous system. Anat Rec B New Anat 2003:271:77-85
Medline   1st Citation  

See J, Zhang, X, Eraydin, N, Mun, SB, Mamontov, P, Golden, JA. Oligodendrocyte maturation is inhibited by bone morphogenetic protein. Mol Cell Neurosci 2004:26:481-92
Crossref   Medline   1st Citation  

Syme R, Bewick, M, Stewart, D, Porter, K, Chadderton, T, Gluck, S. The role of depletion of dimethyl sulfoxide before autografting: on hematologic recovery, side effects, and toxicity. Biol Blood Marrow Transplant 2004:10:135-41
Crossref   Medline   1st Citation   2nd  

Watanabe H, Watanabe, T, Suzuya, H, Wakata, Y, Kaneko, M, Onishi, T. Peripheral blood stem cell mobilization by granulocyte colony-stimulating factor alone and engraftment kinetics following autologous transplantation in children and adolescents with solid tumor. Bone Marrow Transplant 2006:37:661-8
Crossref   Medline   1st Citation  

Weiss S, Reynolds, BA, Vescovi, AL, Morshead, C, Craig, CG, van der Kooy, D. Is there a neural stem cell in the mammalian forebrain? Trends Neurosci 1996:19:387-93
Crossref   Medline   1st Citation  

Xiang Y, Zheng, Q, Jia, B, Huang, G, Xie, C, Pan, J. Ex vivo expansion, adipogenesis and neurogenesis of cryopreserved human bone marrow mesenchymal stem cells. Cell Biol Int 2007:31:444-50
Crossref   Medline   1st Citation  

Yang H, Acker, JP, Cabuhat, M, McGann, LE. Effects of incubation temperature and time after thawing on viability assessment of peripheral hematopoietic progenitor cells cryopreserved for transplantation. Bone Marrow Transplant 2003:32:1021-6
Crossref   Medline   1st Citation  

Zappone MV, Galli, R, Catena, R, Meani, N, De Biasi, S, Mattei, E. Sox2 regulatory sequences direct expression of a (beta)-geo transgene to telencephalic neural stem cells and precursors of the mouse embryo, revealing regionalization of gene expression in CNS stem cells. Development 2000:127:2367-82
Medline   1st Citation  

Zhao J, Hao, HN, Thomas, RL, Lyman, WD. An efficient method for the cryopreservation of fetal human liver hematopoietic progenitor cells. Stem Cells 2001:19:212-8
Crossref   Medline   1st Citation   2nd  


Received 14 December 2007/19 February 2008; accepted 2 April 2008

doi:10.1016/j.cellbi.2008.04.012


ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB)