标题: Cryopreservation Does Not Affect Proliferation and Multipotency of Murine Neural [打印本页] 作者: 江边孤钓 时间: 2009-3-5 10:50 标题: Cryopreservation Does Not Affect Proliferation and Multipotency of Murine Neural
a Department of Neurology, University of Leipzig, Leipzig, Germany; z* I2 H, u2 v' k
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b Department of Neurology, Technical University of Dresden, Dresden, Germany 7 `; c% R |) M0 _% z6 p- _ 3 Q1 o& W( ?- z& @- Y4 D3 eKey Words. Neural precursor cells ? Cryopreservation ? Apoptosis ? Proliferation 7 Q6 z3 L5 K/ s" o 8 l2 ^ t* A8 y& J6 LCorrespondence: 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@medizin.uni-leipzig.de, V7 j/ V4 l) j2 K- L0 N
6 N* @; P0 n7 g: a( R# H; q' kABSTRACT , K. {& }+ e% C4 C1 H7 h4 i+ v8 k: P
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 . The clinical implications of neural stem cells are potentially profound . 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) . However, the role of different cryoprotectants in the preservation of murine NPCs has not been studied in detail.3 ^5 x* t% c6 ?8 g D3 _; D
2 p& W2 l! K' [5 G- [ I8 }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 . Recently, in hematopoietic stem cells (HSCs) and other cell types, the involvement of apoptosis in cryoinjury has been proposed . 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 . Trehalose is effective in cryopreservation, being present either exogenously or endogenously .2 P5 X* O6 @, Z4 u, e
. \/ r3 \$ A8 A. k% AWe 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. 8 C% X# P4 k, E) X: R1 L& j2 v+ D l' O! C, p; V4 J
MATERIALS AND METHODS# Q# ]' y+ o8 J# K& R% ?# R @
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Viability and Apoptosis of Thawed NPCs& W: J& \# m0 d' H' s( b4 Q( |
, @8 Z( f$ a+ R9 O' b# T5 c9 pFor 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. 1 ?9 S, n1 ?2 v& v$ D! H * H3 D& p# ^. _1 _$ P; MFigure 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. 5 O0 K5 U9 d: |+ `! l9 L. t, l. G7 B; ]9 l$ t' Q
As a sensitive probe for the flow cytometric analysis of cells that are in the early stages of apoptosis, we used a fluorochrome-conjugated annexin (annexin–fluorescein isothiocyanate). Frozen-thawed samples were measured during recovery, 24 hours after thawing. As indicated in Figure 1B, apoptotic cell death differed between the samples and remained below 20%. Combination of 10% DMSO and 0.2 M trehalose produced the best effect on survival of NPCs measured within the first 24 hours of recovery. However, freezing medium supplemented with the caspase inhibitor zVAD-fmk did not improve viability of the cells frozen in 10% DMSO (Figs. 1A, 1B). A$ a# |( e4 d! q) s' ?+ \: y. k B # }6 w: o: w) z/ T9 a8 G Z: n& O, D0 REffect of Different Cryoprotectants on Neurosphere Survival Rate ) A+ c; K4 b& F$ d. ]5 z, d* ]* |& I {" ^
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 . 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 # n) T. y3 a, t& Q! O0 K$ r! ?% d' B7 l0 U
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 ( D+ L! t9 x7 \ + g( O; m u3 i5 _& r% o( nIn addition, whole neurospheres or enzymatically dissociated NPCs (treated with Accutase before freezing) were analyzed. The effect of freezing/thawing and cryoprotectants was not different between neurospheres and dissociated NPCs (Fig. 2).5 M1 O+ L2 I I4 j# r- A5 c" }
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Effects of Cryopreservation on Colony Formation ( _% f% o7 |+ O4 v, o, o1 O: `' v3 p3 O6 T
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.+ \) P2 Y9 W9 ~& H! b
% w) O! o% L: t% w U9 I! W6 d6 O8 AFigure 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.2 p6 f! |% _4 ~; p9 [, ~4 G8 l$ m
5 c! c) J0 N& c( w; t, t* o3 R$ x# A3 iEffect of Caspase Inhibitors on Post-Thawing Recovery of NPCs 3 K- h. U* H- S, j' d) l. _) `( a+ K, r ?+ k B4 G
To estimate the effect of caspase inhibitors during freezing process, a broad-spectrum irreversible caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD-fmk) was added to various combinations of cryoprotectants (Fig. 4). During the first week of post-thawing recovery, murine neurospheres died extensively, so in some cases, cell survival was reduced to 9% (Fig. 2; 7-day bars). To address the relevance of apoptosis in recovery of NPCs, the caspase inhibitor zVAD-fmk was added. The relevance of zVAD-fmk was first validated on the post-thawing recovery of the human kidney carcinoma cells (HEK-293T; data not shown). We then applied the same inhibitor on the thawed neurospheres as a supplement to serum-free expansion medium in the case when the cells previously had been frozen in the presence of 10% glycerol. Figure 4 shows that adding this caspase inhibitor significantly improved cell survival (one-way ANOVA; p = .034). All paired multiple-comparison procedures (Student-Newman-Keuls method) revealed that during the first 5 days after thawing, NPC viability was significantly reduced compared with unfrozen controls, whereas an improvement of cell survival was notable already in the presence of 20 μM zVAD-fmk (compared with untreated) and did not significantly change with increased concentrations of the caspase inhibitor. The cells were treated with zVAD-fmk for 2 or 5 days without significant difference on cell survival (data not shown).; @" T8 L" W4 p S: h+ h, _
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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 # V0 c' G" k7 A* S1 _2 b5 w D
' @+ I! W9 k, X& x: H" {Post-Thawing Proliferation and Survival of NPCs* F. y) `5 B [, s5 p
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To assess the proliferative capacity of frozen-thawed neurospheres, PCNA was used as a marker for proliferation. As a marker for cell survival, in some of the thawed samples that continued to grow successfully, the expression of the Bcl-2 protein was evaluated. Both markers were examined after 2 weeks of post-thaw recovery, providing time for proliferation. As presented in Figure 5, samples frozen with 10% DMSO and 10% glycerol seemed to have the highest expression of PCNA and Bcl-2, as quantified by densitometry. ]$ M! v' n0 T& j/ A0 F
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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. - ?8 a, {2 k4 e" a: Q# W* V- D1 t2 a. Q( |
Post-Thawing Differentiation of NPCs" `) {% U; R+ X/ Q
8 \- n# j' Y' a! Y+ r% B" hWithin the first week of the post-thawing period, NPCs formed neurospheres identical to those seen in the fresh tissue (Fig. 6A). After removal of growth factors, cryopreserved and thawed (1-week-old) neurospheres readily differentiated into neurons (?-tubulin III–positive), astrocytes (GFAP-positive), and oligodendrocytes (O4-positive), as detected by immunocytochemistry (Fig. 6B). The relative number of neurons was estimated by calculating the percentage of Tuj1-positive versus DAPI-labeled cells. Although the number of viable cells was markedly reduced during the freezing process, the relative number of neurons in the cryopreserved and thawed tissue was not statistically different from fresh tissue and ranged as follows: 6.12 ± 1.02 in 10% DMSO, 7.64 ± 0.49 in 10% DMSO 10% FCS, 6.88 ± 0.19 in 10% glycerol, and 7.7 ± 1.8 in unfrozen sample.. @$ T' I9 n) o* C/ R; q' w( W6 S5 k
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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, ?III 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.' S) j0 ~! E: |' T3 |
; t: C( P9 g1 d* X; V+ n% @DISCUSSION 0 `5 C% `6 v$ ^0 V4 @3 R5 Y6 x + i5 o9 V" V: \6 KWe 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. 8 f, j) M* h3 y7 d* Z 3 U+ D6 u& E4 L( \! t: h- ^ R* TACKNOWLEDGMENTS/ [( e4 ~$ M8 Z. x( J" s" E
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Sarkar S, Kalia V, Montelaro RC. Caspase-mediated apoptosis and cell death of rhesus macaque CD4 T-cells due to cryopreservation of peripheral blood mononuclear cells can be rescued by cytokine treatment after thawing. Cryobiology 2003;47:44–58. " `& s: x# Z: l( N' C [ ' B( I8 |5 Q; iMatsushita T, Yagi T, Hardin JA et al. Apoptotic cell death and function of cryopreserved porcine hepatocytes in a bioartificial liver. Cell Transplant 2003;12:109–121.(Javorina Milosevica, Alex)作者: 大小年 时间: 2015-6-24 07:52