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Cryopreservation of umbilical cord mesenchymal cells in xenofree conditions
Cytotherapy, 2012; 14: 694–700
Cryopreservation of umbilical cord mesenchymal cells in xenofree conditions
KAREN DE LIMA PRATA1,2, GIL CUNHA DE SANTIS1,2, MARISTELA DELGADO ORELLANA1,2, PATRICIA VIANNA BONINI PALMA1, MARÍA SOL BRASSESCO3 & DIMAS TADEU COVAS1,2 1National
Institute of Science and Technology in Stem Cell and Cell Therapy, Center for Cell Therapy and Regional Blood Center, Ribeirao Preto, Brazil, 2Department of Internal Medicine, School of Medicine, University of Sao Paulo, Ribeirao Preto, Brazil, and 3Department of Pediatrics, School of Medicine, University of Sao Paulo, Ribeirao Preto, Brazil
Abstract Background aims. Mesenchymal stromal cells (MSC) are being used to treat and prevent a variety of clinical conditions. To be readily available, MSC must be cryopreserved until infusion. However, the optimal cryopreservation methods, cryoprotector solutions and MSC sensitivity to dimethyl sulfoxide (DMSO) exposure are unknown. This study investigated these issues. Methods. MSC samples were obtained from human umbilical cord (n ⫽ 15), expanded with Minimal Essential Medium-alpha (α-MEM) 10% human serum (HS), resuspended in 25 mL solution (HS, 10% DMSO, 20% hydroxyethyl starch) and cryopreserved using the BioArchive® system. After a mean of 18 ⫾ 7 days, cell suspensions were thawed and diluted until a DMSO concentration of 2.5% was reached. Samples were tested for cell quantification and viability, immunophenotype and functional assays. Results. Post-thaw cell recovery: 114 ⫾ 2.90% (mean ⫾ SEM). Recovery of viable cells: 93.46 ⫾ 4.41%, 90.17 ⫾ 4.55% and 81.03 ⫾ 4.30% at 30 min, 120 min and 24 h post-thaw, respectively. Cell viability: 89.26 ⫾ 1.56%, 72.71 ⫾ 2.12%, 70.20 ⫾ 2.39% and 63.02 ⫾ 2.33% (P ⬍ 0.0001) pre-cryopreservation and 30 min, 120 min and 24 h post-thaw, respectively. All post-thaw samples had cells that adhered to culture bottles. Post-thaw cell expansion was 4.18 ⫾ 0.17 ⫻, with a doubling time of 38 ⫾ 1.69 h, and their capacity to inhibit peripheral blood mononuclear cells (PBMC) proliferation was similar to that observed before cryopreservation. Differentiation capacity, cell-surface marker profile and cytogenetics were not changed by the cryopreservation procedure. Conclusions. A method for cryopreservation of MSC in bags, in xenofree conditions, is described that facilitates their clinical use. The MSC functional and cytogenetic status and morphologic characteristics were not changed by cryopreservation. It was also demonstrated that MSC are relatively resistant to exposure to DMSO, but we recommend cell infusion as soon as possible. Key Words: cryopreservation, DMSO, MSC, xenofree
Introduction Multipotent mesenchymal stromal cells (MSC) (1) are somatic stem cells, first described by Friedenstein et al. in the 1970s (2), that are located in bone marrow and many other tissues (3). MSC are being used for tissue repair (4), hematopoiesis support (5) and immunomodulation (6). These cells can differentiate into tissues developing from mesoderm, such as bone, cartilage and fat (7), a characteristic that is also explored to define them and evaluate their function in vitro. Other important defining characteristics of MSC are the expression of specific membrane molecules (CD73, CD90 and CD105) together with a lack of expression of hematopoietic markers (CD14, CD34 and CD45),
and their ability to adhere to plastic (8). Besides their potential for differentiation and hematopoiesis support, MSC exert an immunosuppressive effect by a variety of mechanisms (9). Usually, MSC should be expanded ex vivo and cryopreserved before clinical use. However, the optimal protocol for cryopreservation of these cells has not yet been defined. Another important issue is their sensitivity to dimethyl sulfoxide (DMSO) exposure, especially after thawing and before cell infusion, a time lag that can take from minutes to some hours. We designed this study to unveil MSC sensitivity to cryopreservation and different periods of exposure to DMSO after thawing. Furthermore, we
Correspondence: Karen de Lima Prata, Rua Tenente Catao Roxo, 2501 Ribeirão Preto-SP, 14051–140, Brazil. E-mail: [email protected] (Received 31 July 2011; accepted 14 March 2012) ISSN 1465-3249 print/ISSN 1477-2566 online © 2012 Informa Healthcare DOI: 10.3109/14653249.2012.677820
Mesenchymal cell cryopreservation tested a method for cryopreservation and storage of MSC in plastic bags, which facilitates manipulation and clinical use. Methods Isolation and expansion of mesenchymal cells from umbilical cord tissue Human umbilical cords (hUC) were collected (n ⫽ 5) from full-term newborns (39–40 weeks) after Cesarean births, after obtaining informed consent from the person legally responsible for the donor. A piece of hUC, approximately 8 cm, was washed with 1% antibiotic–antimycotic liquid solution (Invitrogen™, Carlsbad, CA, USA) in 0.9% physiologic saline (Baxter, São Paulo, Brazil). A cell suspension was obtained by mechanical disruption followed by enzymatic digestion with a 0.5% collagenase type IA solution (Sigma, Saint Louis, MO, USA) in Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco™, Carlsbad, CA, USA). After incubation at 37°C for 45 min, with frequent agitation, the collagenase solution was inactivated with the addition of RPMI (GibcoBRL, Gaithersburg, MD, USA) and 5% characterized fetal bovine serum (FBS; HyClone™, Logan, UT, USA). The cell-suspension volume was completed to 45 mL with the 1% antibiotic–antimycotic solution on physiologic saline, and homogenized. The biggest tissue fragments were removed by sedimentation. The cell suspension was then centrifuged at 250 g, washed twice, and the cell pellet resuspended in α-MEM (Gibco-BRL) supplemented with 10% allogeneic human serum (HS), 2 mM L-glutamine (Gibco-BRL), 2.4 g/L de Hepes (Gibco-BRL) and 1% antibiotic–antimycotic solution. Next, the cells were plated onto 75-cm2 culture bottles (Greiner Bio-One, Frickenhausen, Germany) and cultured. After 4–7 days, the culture medium was changed and non-adherent cells were removed. The cell culture was maintained with a weekly change of 50% of the medium until cell confluence. At this point, trypsinization was performed with TrypLE® Select® (Invitrogen) for 8 min at 37°C. The cell suspension obtained was centrifuged at 250 g for 10 min, and the cells resuspended in α-MEM supplemented with 10% HS and replated in culture bottles at a concentration of 2–3 ⫻ 103 cells/cm2. The cell cultures were incubated in a humidified atmosphere of 5% CO2 at 37°C. Throughout the entire culture, the cells were analyzed with an Olympus IX71 microscope (Olympus, Tokyo, Japan) equipped with an Olympus DP72 camera. The pooled HS was obtained according to the protocol described by Pytlik et al. (10), with small changes. Briefly, the thawed human AB plasma was transferred to a 2-L bag (plastic bag Viaflex®,
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Baxter), homogenized and mixed with 0.1 M CaCl2 in a 9:1 ratio. The mixture was incubated for 180 min at room temperature and the fibrin clot was removed by filtration through a 170-μm filter. The product was incubated for a further 48 h at 2–6°C and the residual fibrin removed. The serum was sterilized by filtration through a 0.22-μm membrane filter. The aliquots were stored at –30°C until use. Ethical approval was obtained from the Institutional Ethical Review Board (Hospital das Clínicas da Faculdade de Medicina de Ribeirao Preto - Universidade de São Paulo, protocol HCRP number 14906/2010). Cell cryopreservation Cell samples (n ⫽ 15) from between the fifth and seventh passages were detached, washed and resuspended in 25 mL cryopreservation solution (HS, 10% DMSO and 20% hydroxyethyl starch; Plasmin® 460/0.7, 6% solution; HalexIstar, Goiania, Brazil). Cell suspensions were transferred to a 50-mL freezing bag (Thermogenesis®, Rancho Cordova, CA, USA) and cryopreserved on a BioArchive® System (Thermogenesis), according to the BioArchive profile curve, and stored at –196°C. After a mean of 18 ⫾ 7 days in a freezing bag at –196°C, cell suspensions were rapidly thawed in a 37°C water bath (usually 2 min), immediately followed by a stepwise dilution with an equal volume of a cold thawing solution [20% HS, 5% acid citrate dextrose (ACD) anticoagulant formula A (JP Indústria Farmacêutica SA, Ribeirão Preto, Brazil) in 0.9% physiologic saline] under gentle shaking at room temperature. After an equilibrium time of 2 min, the cell suspension was diluted with two volumes of the thawing solution (final dilution of 1/4). The diluted cell suspension was transferred to a 300-mL dry plastic bag (Fresenius Medical Care LTDA, Jaguariúna, Brazil). The transference bag with the cell suspension was stored in a blood bank refrigerator (2–6°C). A sample was collected from each cell suspension for quantification and cultivation in 75-cm2 culture bottles, for immunophenotyping functional assays and cytogenetics. After 2–4 days of thawing, another sample was collected and plated onto 25-cm2 culture bottles to assess the capability of the cells to adhere. Flow cytometry assay The immunophenotype and cell viability were evaluated by flow cytometry on a FACSCalibur™ cytometer (Becton Dickinson) and analyzed with CELLQuest™ software (Becton Dickinson, San Jose, CA, USA) as described previously (11). For the cell viability assay, 105 cells collected before freezing and 30 min, 120 min and 24 h after thawing were labeled with propidium iodide (PI; Sigma-Aldrich, Saint Louis, MO, EUA)
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and the Annexin V Apoptosis Detection Kit I (BDPharmingen, San Jose, CA, USA) according to the manufacturer’s protocol. Viable cells were double negative. Cell samples (n ⫽ 10), obtained immediately after thawing and after cultivation, were labeled with CD14– phycoerythrin (PE), CD45–fluorescein isothiocyanate (FITC), CD34–peridinin chlorophyll (PerCP), CD31– FITC, HLA-DR–FITC, CD338–PE, CD90–PE, CD73–PE, CD105–PE, CD146–PE, CD166–PE, CD44–FITC, HLA-ABC–PE, CD49e–PE, CD13– allophycocyanin (APC), CD29–APC and CD54–PE (BD Pharmingen). Growth kinetics Population doubling (PD) was calculated between the first and second passages after thawing with the formula logn/log2, as described by Stenderup et al. (12), where n is the number of cells counted at the time of trypsinization divided by the initial number of cells plated. The doubling time was calculated by dividing the number of hours between these passages by the cell expansion during the same period. Functional tests Determination of cell immunomodulatory properties. In vitro immunomodulatory properties of umbilical cord (UC) MSC were assessed by co-culture with carboxyfluorescein diacetate succinimidyl ester (CFSE; Molecular Probes®; Invitrogen)-labeled normal peripheral blood mononuclear cells (PBMC). These assays were performed on five cell samples before (between the third and fifth passages) and after (cells on the first passage after thawing of the cells cryopreserved on the seventh passage) cryopreservation. The UC MSC were seeded at a density of 2.5 ⫻ 105, 1.0 ⫻ 105 and 0.5 ⫻ 105/well in 24-well cell culture plates. Once the cells had attached to the surface (approximately 4 h), 5 ⫻ 105 CFSE-stained PBMC were transferred to the well. PBMC proliferations were stimulated using 5 μL phytohemagglutinin (PHA; 0.25 μg/mL; Sigma). When cells marked with CFSE divide, the CFSE is distributed equally between the daughter cells, so each daughter cell presents half of the green fluorescence intensity of the mother cell. Therefore, the loss of cell fluorescence is proportional to the cell proliferation rate. Cells were harvested after 5 days of co-cultivation (37°C with 5% CO2) to assess normal lymphocyte proliferation based on CFSE intensity by flow cytometry after labeling with anti-CD3–APC (15 000 events were acquired). For the analysis, the proliferating population of cells was gated (R1) in lymphocytes that were CD3⫹ , and the events represented as a fluorescence histogram (FL1–CFSE). As assay controls, non-labeled
PBMC and CFSE-labeled PBMC, stimulated or not with PHA and cultivated without UC MSC as a feeder layer, were used. All experiments were performed in triplicates. An example of flow cytometry analysis of the proliferating T lymphocytes in PBMC can be seen in Supplementary Figure 1 (online only, available at http://www.informahealthcare.com/doi/ abs/10.3109/14653249.2012.677820). The percentage of inhibition was calculated with the formula [(A – B)/A] ⫻ 100, where A corresponds to CFSE-labeled PBMC stimulated with PHA proliferation minus CFSE-labeled PBMC (not stimulated with PHA) proliferation, both cultivated without UC MSC as a feeder layer, and B corresponds to the mean proliferation percentage of CFSE-labeled PBMC stimulated with PHA and cultivated with UC MSC as a feeder layer under each condition. Differentiation into adipocytes and osteocytes. The UC MSC capacity for differentiation into adipocytes and osteocytes was evaluated as described previously (13). Briefly, three cell samples on the first passage after thawing (they were cryopreserved on the seventh passage) were incubated with the specific differentiation medium, fixed and stained using the von Kossa method (for calcium deposition) or with Sudan II and Scarlet stains (for fat accumulation). Cells were analyzed with a model Axioskop 2.0 Zeiss microscope (Carl Zeiss, Oberkochen, Germany) equipped with an Axiocam camera (Zeiss). Cytogenetic studies. The cytogenetic status after cryopreservation was evaluated by G-banding in three samples from the seventh passage. UC MSC were cultivated in 75-cm2 culture bottles until reaching a confluence of 70%, when the cycle cell was interrupted with Colcemid® (0.15 μg/mL; SigmaAldrich). Cells were then incubated (37°C with 5% CO2) for 6 h and trypsinized. The cell suspension obtained was centrifuged at 450 g for 7 min, and the cellular pellet resuspended in KCl (75 mM, 37°C; Merck, Darmstadt, Germany), incubated (37°C, humid) for 20 min, centrifuged again and resuspended in a methanol/acetic acid (Merck) solution at a proportion of 3:1. The cells were washed with this solution at least three times. Cytogenetic analysis was then performed by G-banding (50 metaphases) and the results interpreted according to the International System for Human Cytogenetics Nomenclature 2005 (14). Additionally, 100 Giemsa-stained metaphases were screened for aberrations that would disrupt chromosome integrity, such as gaps, breaks, fragments, and other structural abnormalities, such as rings and dicentrics.
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Figure 1. Cell viability evaluation pre-cryopreservation and postthaw. Double-negative cells for PI and Annexin V were considered to be viable cells. (A) Total and viable cell quantification, precryopreservation and post-thaw. (B) Flow cytometer viable cell quantification. Straight line, mean; ∗∗∗P ⬍ 0.001; ∗∗P ⬍ 0.01 (repeated-measures ANOVA).
Statistical analyzes. The data were reported as mean (⫾ SEM) or median (range). Paired t-tests, Wilcoxon matched-pairs signed rank tests and repeated-measures ANOVA with Tukey post-tests were applied, as appropriate, using GraphPad InStat software, version 3.0 for Windows (GraphPad Software, San Diego, CA, USA; www.graphpad.com). We assumed a level of significance of P ⬍ 0.05. Results A total of 15 UC MSC was submitted to cryopreservation tests. Three additional samples were cryopreserved for cytogenetics and differentiation analysis. The mean ⫾ SEM of the total cell quantification pre-cryopreservation and post-thaw was, respectively, 8.7 ⫾ 0.72 ⫻ 107 and 9.7 ⫾ 0.72 ⫻ 107, a with post-thaw cell recuperation of 114 ⫾ 2.90%. The absolute number of viable cells (⫻ 107) pre-cryopreservation and 30 min, 120 min and 24 h
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post-thaw was, respectively, 7.77 ⫾ 0.68, 7.07 ⫾ 0.55, 6.81 ⫾ 0.53 and 6.11 ⫾ 0.48 cells (P ⬍ 0.0001, repeated-measures ANOVA), with a difference between the groups of viable cells pre-cryopreservation and 120 min post-thaw (P ⬍ 0.01), viable cells precryopreservation and 24 h post-thaw (P ⬍ 0.001) and viable cells 30 min and 24 h post-thaw (P ⬍ 0.01). The viable cell recuperation 30 min, 120 min and 24 h post-thaw was, respectively, 93.46 ⫾ 4.41%, 90.17 ⫾ 4.55% and 81.03 ⫾ 4.30% (Figure 1A). The percentage of viable cell pre-cryopreservation and 30 min, 120 min and 24 h post-thaw was, respectively, 89.26 ⫾ 1.56, 72.71 ⫾ 2.12, 70.20 ⫾ 2.39 and 63.02 ⫾ 2.33 (P ⬍ 0.0001), with a difference between all groups except cell viability at 30 and 120 min post-thaw (Figure 1B). Cell aliquots of 2–5 ⫻ 105 were plated in 75-cm2 culture bottles for functional assay and cell expansion evaluation post-thaw. Cells from all 15 samples adhered to the culture bottles. Cell expansion between the first and second passages post-thaw was 4.18 ⫾ 0.17 ⫻, with a doubling time of 38.06 ⫾ 1.69 h. Fourteen samples were stored in a blood bank refrigerator, in transference bags (2.5% DMSO), for 73 (36–97) h, when 500 μL were collected and plated in 25-cm2 culture bottles. Cells from all samples adhered to the bottles and expanded, preserving their morphologic characteristics (Figure 2). The UC MSC surface marker profiles (n ⫽ 10) from samples analyzed immediately after thawing and post-cultivation were similar. The cells were negative for endothelial, hematopoietic and HLA-DR surface markers and positive for mesenchymal (CD90, CD73, CD105, CD166 and CD146), adhesion molecule (CD44, CD49e, CD29, CD13 and CD54) and HLA-ABC surface markers. There was a difference between only two markers, CD73 (P ⬍ 0.002, Wilcoxon matched-pairs signed-ranks test) and CD146 (P ⬍ 0.0488) (Table I). Pre-cryopreservation results can be seen in Supplementary Table I (online only, available at http://www.informahealthcare.com/doi/ abs/10.3109/14653249.2012.677820). The samples thawed in the seventh passage (n ⫽ 5) where tested for their capacity to inhibit PBMC proliferation. The results obtained in the eight passage (the first post-thaw) were compared with the same samples tested pre-cryopreservation (third to fifth passages). The medians (ranges) of the percentage inhibition of cell proliferation pre-cryopreservation and post-thaw were, at a concentration of 1:2 (PBMC:MSC), 85.1 (70.69–92.78) and 89.31 (70.66–93.55) (P ⫽ 0.6250, Wilcoxon matched-pairs signed-rank test), at a concentration of 1:5, 72.16 (50.27–83.57) and 76.81 (40.65–86.57) (P ⫽ 0.4375), and at a concentration of 1:10, 54.74 (42.89–78.86) and 68.4 (32.00–78.10) (P ⫽ 0.4375) (Figure 3).
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K. de Lima Prata et al. Table I. Mesenchymal cell immunophenotype.a Marker CD14 CD45 CD34 CD31 HLA-DR CD338 CD90 CD731 CD105 CD1461 CD166 CD44 HLA-ABC CD49e CD29 CD13 CD54
Post-thaw
Post-cultivation
0.05 0.03 0.27 0.23 0.10 0.67 99.03 89.44 95.34 77.79 90.19 95.76 88.05 96.75 98.98 97.72 93.36
0.03 0.08 0.93 0.63 0.25 1.35 98.02 94.68 96.42 86.95 93.56 95.52 86.46 97.00 99.32 98.96 91.39
samples, except for CD338 (n ⫽ 9), were evaluated for each marker, immediately post-thaw and after cultivation. Values represent the median of the percentage of cells positive for the expression of each marker gated according to the appropriate isotype control. 1Samples with P ⬍ 0.05 (Wilcoxon matched-pairs signed-rank test). aTen
Figure 2. Post-thaw morphologic characterization of UC MSC. (A–C) UC MSC plated onto 25-cm2 culture bottles 72 h after thawing in the sixth passage. (A) 40 ⫻, (B) 100 ⫻, (C) 200 ⫻.
The UC MSC from all samples tested (n ⫽ 3) were able to differentiate into adipocytes and osteocytes (Figure 4) and showed a normal karyotype by cytogenetic analysis (G-banding of samples) (Table II). Discussion We have described a cryopreservation method for UC MSC in bags in xenofree conditions. Furthermore, we have shown that UC MSC are relatively resistant to DMSO exposure after thawing, a common event in the context of cell therapy.
We employed the BioArchive® system to cryopreserve the UC MSC because it is a computerized device that was developed to prevent exposure of the frozen cord blood units to changes in the temperature gradient. Moreover, it allows automated freezing and individually controlled storage of each unit, which has improved recovery and viability of hematopoietic stem cells after thawing (15). Cell recovery after thawing was excellent (114%), which means that virtually no cells were lost during cryopreservation, storage and the thaw/ dilution procedures. The mean recovery of viable cells 30 min after thawing was 73%. This result is better than those obtained by others (63%) (16). In addition, the method we used (PI ⫹ Annexin V, flow cytometry) to evaluate cell viability is more accurate than the most frequently used trypan blue (16–18). As expected, there was a progressive decrease of cell viability after thawing. This fact suggests that UC MSC are damaged by DMSO exposure and supports our decision to infuse the cells as soon as possible, preferably within the first 2 h after thawing. It is intuitive to assume a certain degree of cell loss (whichever cell) when exposed to DMSO; however, regarding UC MSC, we have shown the magnitude of this phenomenon. Besides cell viability, other parameters are important for evaluating UC MSC integrity, such as their immunophenotypic profile and function. We found that markers CD73 and CD146 had a decreased expression just after thawing, but
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Figure 3. UC MSC inhibition of PBMC proliferation precryopreservation and post-thaw. PBMC were polyclonally stimulated with PHA during co-cultivation with different concentrations of five UC MSC samples pre-cryopreservation (third to fifth passages) and post-thaw and cultivation (eight passage). There were no differences between the groups (P ⬎ 0.05, Wilcoxon matched-pairs test). The graphic is plotted with median and range values. The ratios are of PBMC:UC MSC.
recovered their original expression pattern when the cells were again put to cultivation. This finding suggests that there is a transitory decrease of these markers with the cryopreservation–thawing–dilution process. Probably this loss of expression, besides being transitory, does not interfere with the cell function, as the cells adhered to the culture bottle. The loss of expression of markers secondary to the cryopreservation–thawing process is not surprising. Sarugaser et al. (16) have shown that the HLA-ABC expression was stable only until the fifth passage and then lost with the cryopreservation– thawing process. However, this specific finding was not confirmed in our study, where we have shown a stronger expression of this marker in samples evaluated between the fifth and seventh passages, immediately post-thaw and after culture expansion. Most importantly, UC MSC preserved their immunomodulatory properties, despite having been submitted to two different situations that could affected their potency: the cryopreservation–thawing process, and the discrepancy in the passage (third to fifth pre-cryopreservation versus eighth post-thaw and cultivation). Also, the cryopreservation–thawing process did not alter the UC MSC differentiation
Figure 4. UC MSC differentiated into adipocytes and osteocytes post-cryopreservation. Example of a UC MSC sample differentiated into adipocytes (A; Sudan II scarlet and Harris hematoxylin staining; 630 ⫻) and osteocytes (B; von Kossa and Harris hematoxylin staining; 630 ⫻) on the 28th day of culture.
ability, or chromosome integrity, as observed by cytogenetic analysis. In summary, we think that the method described here is efficient for UC MSC cryopreservation, especially the employment of plastic bags, which facilitates their clinical use. Moreover, UC MSC are relatively resistant to DMSO exposure, but there is some decrease in viability with progressive exposure. For this reason we recommend cell infusion as soon as possible, preferably within the first 2 h of thawing.
Table II. Cytogenetic analysis of mesenchymal cells after cryopreservation. Sample UC MSC-06 UC MSC-07 UC MSC-08 aOne-hundred
Passage
Karyotype (G-band)
Structural aberrationsa
8 8 8
46, XY (50) 46, XY (50) 46, XY (50)
Not observed Not observed Not observed
conventionally stained metaphases were screened for chromatidic and chromosome gaps, and breaks, fragments, double minutes and other aberrations such as rings and dicentrics.
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Acknowledgements The authors thank Karina S. Candido, Taisa R. Fernandes, Samia R. Caruso and Camila C. O. M. Bonaldo for the laboratory assistance, and Maria Fernanda Capeli for the umbilical cord collection. They also would like to thank to CEI (Comércio Exportação e Importação de materiais médicos LTDA) for the cryopreservation bag donation. Disclosure of interests: The authors declare no conflict of interest. References 1. Horwitz EM, Le Blanc K, Dominici M, Mueller I, SlaperCortenbach I, Marini FC, et al. Clarification of the nomenclature for MSC: The International Society for Cellular Therapy position statement. Cytotherapy. 2005;7:393–5. 2. Friedenstein AJ, Piatetzky S II, Petrakova KV. Osteogenesis in transplants of bone marrow cells. J Embryol Exp Morphol. 1966;16:381–90. 3. Covas DT, Panepucci RA, Fontes AM, Silva WA Jr, Orellana MD, Freitas MC, et al. Multipotent mesenchymal stromal cells obtained from diverse human tissues share functional properties and gene-expression profile with CD146 ⫹ perivascular cells and fibroblasts. Exp Hematol. 2008;36:642–54. 4. Ringden O, Uzunel M, Sundberg B, Lonnies L, Nava S, Gustafsson J, et al. Tissue repair using allogeneic mesenchymal stem cells for hemorrhagic cystitis, pneumomediastinum and perforated colon. Leukemia. 2007;21:2271–6. 5. Le Blanc K, Samuelsson H, Gustafsson B, Remberger M, Sundberg B, Arvidson J, et al. Transplantation of mesenchymal stem cells to enhance engraftment of hematopoietic stem cells. Leukemia. 2007;21:1733–8. 6. Le Blanc K, Frassoni F, Ball L, Locatelli F, Roelofs H, Lewis I, et al. Mesenchymal stem cells for treatment of steroid-resistant, severe, acute graft-versus-host disease: a phase II study. Lancet. 2008;371:1579–86.
Supplementary material available online Supplementary Figure 1 and Table I.
7. Prockop DJ. Marrow stromal cells as stem cells for nonhematopoietic tissues. Science. 1997;276:71–4. 8. Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini F, Krause D, et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy. 2006;8:315–7. 9. Nauta AJ, Fibbe WE. Immunomodulatory properties of mesenchymal stromal cells. Blood. 2007;110:3499–506. 10. Pytlik R, Stehlik D, Soukup T, Kalbacova M, Rypacek F, Trc T, et al. The cultivation of human multipotent mesenchymal stromal cells in clinical grade medium for bone tissue engineering. Biomaterials. 2009;30:3415–27. 11. Silva WA Jr, Covas DT, Panepucci RA, Proto-Siqueira R, Siufi JL, Zanette DL, et al. The profile of gene expression of human marrow mesenchymal stem cells. Stem Cells. 2003;21: 661–9. 12. Stenderup K, Justesen J, Clausen C, Kassem M. Aging is associated with decreased maximal life span and accelerated senescence of bone marrow stromal cells. Bone. 2003;33: 919–26. 13. Covas DT, Siufi JL, Silva AR, Orellana MD. Isolation and culture of umbilical vein mesenchymal stem cells. Braz J Med Biol Res. 2003;36:1179–83. 14. Shaffer LG, Tommerup N. ISCN 2005: An International System for Human Cytogenetic Nomenclature. Basel: S. Karger; 2005. 15. Rubinstein P. Cord blood banking for clinical transplantation. Bone Marrow Transplant. 2009;44:635–42. 16. Sarugaser R, Lickorish D, Baksh D, Hosseini MM, Davies JE. Human umbilical cord perivascular (HUCPV) cells: a source of mesenchymal progenitors. Stem Cells. 2005;23: 220–9. 17. Samuelsson H, Ringden O, Lonnies H, Le Blanc K. Optimizing in vitro conditions for immunomodulation and expansion of mesenchymal stromal cells. Cytotherapy. 2009; 11:129–36. 18. Chin SP, Poey AC, Wong CY, Chang SK, Teh W, Mohr TJ, et al. Cryopreserved mesenchymal stromal cell treatment is safe and feasible for severe dilated ischemic cardiomyopathy. Cytotherapy. 2010;12:31–7.
Cryopreservation of umbilical cord mesenchymal cells in xenofree conditions
KAREN DE LIMA PRATA1,2, GIL CUNHA DE SANTIS1,2, MARISTELA DELGADO ORELLANA1,2, PATRICIA VIANNA BONINI PALMA1, MARÍA SOL BRASSESCO3 & DIMAS TADEU COVAS1,2 1National
Institute of Science and Technology in Stem Cell and Cell Therapy, Center for Cell Therapy and Regional Blood Center, Ribeirao Preto, Brazil, 2Department of Internal Medicine, School of Medicine, University of Sao Paulo, Ribeirao Preto, Brazil, and 3Department of Pediatrics, School of Medicine, University of Sao Paulo, Ribeirao Preto, Brazil
Abstract Background aims. Mesenchymal stromal cells (MSC) are being used to treat and prevent a variety of clinical conditions. To be readily available, MSC must be cryopreserved until infusion. However, the optimal cryopreservation methods, cryoprotector solutions and MSC sensitivity to dimethyl sulfoxide (DMSO) exposure are unknown. This study investigated these issues. Methods. MSC samples were obtained from human umbilical cord (n ⫽ 15), expanded with Minimal Essential Medium-alpha (α-MEM) 10% human serum (HS), resuspended in 25 mL solution (HS, 10% DMSO, 20% hydroxyethyl starch) and cryopreserved using the BioArchive® system. After a mean of 18 ⫾ 7 days, cell suspensions were thawed and diluted until a DMSO concentration of 2.5% was reached. Samples were tested for cell quantification and viability, immunophenotype and functional assays. Results. Post-thaw cell recovery: 114 ⫾ 2.90% (mean ⫾ SEM). Recovery of viable cells: 93.46 ⫾ 4.41%, 90.17 ⫾ 4.55% and 81.03 ⫾ 4.30% at 30 min, 120 min and 24 h post-thaw, respectively. Cell viability: 89.26 ⫾ 1.56%, 72.71 ⫾ 2.12%, 70.20 ⫾ 2.39% and 63.02 ⫾ 2.33% (P ⬍ 0.0001) pre-cryopreservation and 30 min, 120 min and 24 h post-thaw, respectively. All post-thaw samples had cells that adhered to culture bottles. Post-thaw cell expansion was 4.18 ⫾ 0.17 ⫻, with a doubling time of 38 ⫾ 1.69 h, and their capacity to inhibit peripheral blood mononuclear cells (PBMC) proliferation was similar to that observed before cryopreservation. Differentiation capacity, cell-surface marker profile and cytogenetics were not changed by the cryopreservation procedure. Conclusions. A method for cryopreservation of MSC in bags, in xenofree conditions, is described that facilitates their clinical use. The MSC functional and cytogenetic status and morphologic characteristics were not changed by cryopreservation. It was also demonstrated that MSC are relatively resistant to exposure to DMSO, but we recommend cell infusion as soon as possible. Key Words: cryopreservation, DMSO, MSC, xenofree
Introduction Multipotent mesenchymal stromal cells (MSC) (1) are somatic stem cells, first described by Friedenstein et al. in the 1970s (2), that are located in bone marrow and many other tissues (3). MSC are being used for tissue repair (4), hematopoiesis support (5) and immunomodulation (6). These cells can differentiate into tissues developing from mesoderm, such as bone, cartilage and fat (7), a characteristic that is also explored to define them and evaluate their function in vitro. Other important defining characteristics of MSC are the expression of specific membrane molecules (CD73, CD90 and CD105) together with a lack of expression of hematopoietic markers (CD14, CD34 and CD45),
and their ability to adhere to plastic (8). Besides their potential for differentiation and hematopoiesis support, MSC exert an immunosuppressive effect by a variety of mechanisms (9). Usually, MSC should be expanded ex vivo and cryopreserved before clinical use. However, the optimal protocol for cryopreservation of these cells has not yet been defined. Another important issue is their sensitivity to dimethyl sulfoxide (DMSO) exposure, especially after thawing and before cell infusion, a time lag that can take from minutes to some hours. We designed this study to unveil MSC sensitivity to cryopreservation and different periods of exposure to DMSO after thawing. Furthermore, we
Correspondence: Karen de Lima Prata, Rua Tenente Catao Roxo, 2501 Ribeirão Preto-SP, 14051–140, Brazil. E-mail: [email protected] (Received 31 July 2011; accepted 14 March 2012) ISSN 1465-3249 print/ISSN 1477-2566 online © 2012 Informa Healthcare DOI: 10.3109/14653249.2012.677820
Mesenchymal cell cryopreservation tested a method for cryopreservation and storage of MSC in plastic bags, which facilitates manipulation and clinical use. Methods Isolation and expansion of mesenchymal cells from umbilical cord tissue Human umbilical cords (hUC) were collected (n ⫽ 5) from full-term newborns (39–40 weeks) after Cesarean births, after obtaining informed consent from the person legally responsible for the donor. A piece of hUC, approximately 8 cm, was washed with 1% antibiotic–antimycotic liquid solution (Invitrogen™, Carlsbad, CA, USA) in 0.9% physiologic saline (Baxter, São Paulo, Brazil). A cell suspension was obtained by mechanical disruption followed by enzymatic digestion with a 0.5% collagenase type IA solution (Sigma, Saint Louis, MO, USA) in Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco™, Carlsbad, CA, USA). After incubation at 37°C for 45 min, with frequent agitation, the collagenase solution was inactivated with the addition of RPMI (GibcoBRL, Gaithersburg, MD, USA) and 5% characterized fetal bovine serum (FBS; HyClone™, Logan, UT, USA). The cell-suspension volume was completed to 45 mL with the 1% antibiotic–antimycotic solution on physiologic saline, and homogenized. The biggest tissue fragments were removed by sedimentation. The cell suspension was then centrifuged at 250 g, washed twice, and the cell pellet resuspended in α-MEM (Gibco-BRL) supplemented with 10% allogeneic human serum (HS), 2 mM L-glutamine (Gibco-BRL), 2.4 g/L de Hepes (Gibco-BRL) and 1% antibiotic–antimycotic solution. Next, the cells were plated onto 75-cm2 culture bottles (Greiner Bio-One, Frickenhausen, Germany) and cultured. After 4–7 days, the culture medium was changed and non-adherent cells were removed. The cell culture was maintained with a weekly change of 50% of the medium until cell confluence. At this point, trypsinization was performed with TrypLE® Select® (Invitrogen) for 8 min at 37°C. The cell suspension obtained was centrifuged at 250 g for 10 min, and the cells resuspended in α-MEM supplemented with 10% HS and replated in culture bottles at a concentration of 2–3 ⫻ 103 cells/cm2. The cell cultures were incubated in a humidified atmosphere of 5% CO2 at 37°C. Throughout the entire culture, the cells were analyzed with an Olympus IX71 microscope (Olympus, Tokyo, Japan) equipped with an Olympus DP72 camera. The pooled HS was obtained according to the protocol described by Pytlik et al. (10), with small changes. Briefly, the thawed human AB plasma was transferred to a 2-L bag (plastic bag Viaflex®,
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Baxter), homogenized and mixed with 0.1 M CaCl2 in a 9:1 ratio. The mixture was incubated for 180 min at room temperature and the fibrin clot was removed by filtration through a 170-μm filter. The product was incubated for a further 48 h at 2–6°C and the residual fibrin removed. The serum was sterilized by filtration through a 0.22-μm membrane filter. The aliquots were stored at –30°C until use. Ethical approval was obtained from the Institutional Ethical Review Board (Hospital das Clínicas da Faculdade de Medicina de Ribeirao Preto - Universidade de São Paulo, protocol HCRP number 14906/2010). Cell cryopreservation Cell samples (n ⫽ 15) from between the fifth and seventh passages were detached, washed and resuspended in 25 mL cryopreservation solution (HS, 10% DMSO and 20% hydroxyethyl starch; Plasmin® 460/0.7, 6% solution; HalexIstar, Goiania, Brazil). Cell suspensions were transferred to a 50-mL freezing bag (Thermogenesis®, Rancho Cordova, CA, USA) and cryopreserved on a BioArchive® System (Thermogenesis), according to the BioArchive profile curve, and stored at –196°C. After a mean of 18 ⫾ 7 days in a freezing bag at –196°C, cell suspensions were rapidly thawed in a 37°C water bath (usually 2 min), immediately followed by a stepwise dilution with an equal volume of a cold thawing solution [20% HS, 5% acid citrate dextrose (ACD) anticoagulant formula A (JP Indústria Farmacêutica SA, Ribeirão Preto, Brazil) in 0.9% physiologic saline] under gentle shaking at room temperature. After an equilibrium time of 2 min, the cell suspension was diluted with two volumes of the thawing solution (final dilution of 1/4). The diluted cell suspension was transferred to a 300-mL dry plastic bag (Fresenius Medical Care LTDA, Jaguariúna, Brazil). The transference bag with the cell suspension was stored in a blood bank refrigerator (2–6°C). A sample was collected from each cell suspension for quantification and cultivation in 75-cm2 culture bottles, for immunophenotyping functional assays and cytogenetics. After 2–4 days of thawing, another sample was collected and plated onto 25-cm2 culture bottles to assess the capability of the cells to adhere. Flow cytometry assay The immunophenotype and cell viability were evaluated by flow cytometry on a FACSCalibur™ cytometer (Becton Dickinson) and analyzed with CELLQuest™ software (Becton Dickinson, San Jose, CA, USA) as described previously (11). For the cell viability assay, 105 cells collected before freezing and 30 min, 120 min and 24 h after thawing were labeled with propidium iodide (PI; Sigma-Aldrich, Saint Louis, MO, EUA)
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and the Annexin V Apoptosis Detection Kit I (BDPharmingen, San Jose, CA, USA) according to the manufacturer’s protocol. Viable cells were double negative. Cell samples (n ⫽ 10), obtained immediately after thawing and after cultivation, were labeled with CD14– phycoerythrin (PE), CD45–fluorescein isothiocyanate (FITC), CD34–peridinin chlorophyll (PerCP), CD31– FITC, HLA-DR–FITC, CD338–PE, CD90–PE, CD73–PE, CD105–PE, CD146–PE, CD166–PE, CD44–FITC, HLA-ABC–PE, CD49e–PE, CD13– allophycocyanin (APC), CD29–APC and CD54–PE (BD Pharmingen). Growth kinetics Population doubling (PD) was calculated between the first and second passages after thawing with the formula logn/log2, as described by Stenderup et al. (12), where n is the number of cells counted at the time of trypsinization divided by the initial number of cells plated. The doubling time was calculated by dividing the number of hours between these passages by the cell expansion during the same period. Functional tests Determination of cell immunomodulatory properties. In vitro immunomodulatory properties of umbilical cord (UC) MSC were assessed by co-culture with carboxyfluorescein diacetate succinimidyl ester (CFSE; Molecular Probes®; Invitrogen)-labeled normal peripheral blood mononuclear cells (PBMC). These assays were performed on five cell samples before (between the third and fifth passages) and after (cells on the first passage after thawing of the cells cryopreserved on the seventh passage) cryopreservation. The UC MSC were seeded at a density of 2.5 ⫻ 105, 1.0 ⫻ 105 and 0.5 ⫻ 105/well in 24-well cell culture plates. Once the cells had attached to the surface (approximately 4 h), 5 ⫻ 105 CFSE-stained PBMC were transferred to the well. PBMC proliferations were stimulated using 5 μL phytohemagglutinin (PHA; 0.25 μg/mL; Sigma). When cells marked with CFSE divide, the CFSE is distributed equally between the daughter cells, so each daughter cell presents half of the green fluorescence intensity of the mother cell. Therefore, the loss of cell fluorescence is proportional to the cell proliferation rate. Cells were harvested after 5 days of co-cultivation (37°C with 5% CO2) to assess normal lymphocyte proliferation based on CFSE intensity by flow cytometry after labeling with anti-CD3–APC (15 000 events were acquired). For the analysis, the proliferating population of cells was gated (R1) in lymphocytes that were CD3⫹ , and the events represented as a fluorescence histogram (FL1–CFSE). As assay controls, non-labeled
PBMC and CFSE-labeled PBMC, stimulated or not with PHA and cultivated without UC MSC as a feeder layer, were used. All experiments were performed in triplicates. An example of flow cytometry analysis of the proliferating T lymphocytes in PBMC can be seen in Supplementary Figure 1 (online only, available at http://www.informahealthcare.com/doi/ abs/10.3109/14653249.2012.677820). The percentage of inhibition was calculated with the formula [(A – B)/A] ⫻ 100, where A corresponds to CFSE-labeled PBMC stimulated with PHA proliferation minus CFSE-labeled PBMC (not stimulated with PHA) proliferation, both cultivated without UC MSC as a feeder layer, and B corresponds to the mean proliferation percentage of CFSE-labeled PBMC stimulated with PHA and cultivated with UC MSC as a feeder layer under each condition. Differentiation into adipocytes and osteocytes. The UC MSC capacity for differentiation into adipocytes and osteocytes was evaluated as described previously (13). Briefly, three cell samples on the first passage after thawing (they were cryopreserved on the seventh passage) were incubated with the specific differentiation medium, fixed and stained using the von Kossa method (for calcium deposition) or with Sudan II and Scarlet stains (for fat accumulation). Cells were analyzed with a model Axioskop 2.0 Zeiss microscope (Carl Zeiss, Oberkochen, Germany) equipped with an Axiocam camera (Zeiss). Cytogenetic studies. The cytogenetic status after cryopreservation was evaluated by G-banding in three samples from the seventh passage. UC MSC were cultivated in 75-cm2 culture bottles until reaching a confluence of 70%, when the cycle cell was interrupted with Colcemid® (0.15 μg/mL; SigmaAldrich). Cells were then incubated (37°C with 5% CO2) for 6 h and trypsinized. The cell suspension obtained was centrifuged at 450 g for 7 min, and the cellular pellet resuspended in KCl (75 mM, 37°C; Merck, Darmstadt, Germany), incubated (37°C, humid) for 20 min, centrifuged again and resuspended in a methanol/acetic acid (Merck) solution at a proportion of 3:1. The cells were washed with this solution at least three times. Cytogenetic analysis was then performed by G-banding (50 metaphases) and the results interpreted according to the International System for Human Cytogenetics Nomenclature 2005 (14). Additionally, 100 Giemsa-stained metaphases were screened for aberrations that would disrupt chromosome integrity, such as gaps, breaks, fragments, and other structural abnormalities, such as rings and dicentrics.
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Figure 1. Cell viability evaluation pre-cryopreservation and postthaw. Double-negative cells for PI and Annexin V were considered to be viable cells. (A) Total and viable cell quantification, precryopreservation and post-thaw. (B) Flow cytometer viable cell quantification. Straight line, mean; ∗∗∗P ⬍ 0.001; ∗∗P ⬍ 0.01 (repeated-measures ANOVA).
Statistical analyzes. The data were reported as mean (⫾ SEM) or median (range). Paired t-tests, Wilcoxon matched-pairs signed rank tests and repeated-measures ANOVA with Tukey post-tests were applied, as appropriate, using GraphPad InStat software, version 3.0 for Windows (GraphPad Software, San Diego, CA, USA; www.graphpad.com). We assumed a level of significance of P ⬍ 0.05. Results A total of 15 UC MSC was submitted to cryopreservation tests. Three additional samples were cryopreserved for cytogenetics and differentiation analysis. The mean ⫾ SEM of the total cell quantification pre-cryopreservation and post-thaw was, respectively, 8.7 ⫾ 0.72 ⫻ 107 and 9.7 ⫾ 0.72 ⫻ 107, a with post-thaw cell recuperation of 114 ⫾ 2.90%. The absolute number of viable cells (⫻ 107) pre-cryopreservation and 30 min, 120 min and 24 h
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post-thaw was, respectively, 7.77 ⫾ 0.68, 7.07 ⫾ 0.55, 6.81 ⫾ 0.53 and 6.11 ⫾ 0.48 cells (P ⬍ 0.0001, repeated-measures ANOVA), with a difference between the groups of viable cells pre-cryopreservation and 120 min post-thaw (P ⬍ 0.01), viable cells precryopreservation and 24 h post-thaw (P ⬍ 0.001) and viable cells 30 min and 24 h post-thaw (P ⬍ 0.01). The viable cell recuperation 30 min, 120 min and 24 h post-thaw was, respectively, 93.46 ⫾ 4.41%, 90.17 ⫾ 4.55% and 81.03 ⫾ 4.30% (Figure 1A). The percentage of viable cell pre-cryopreservation and 30 min, 120 min and 24 h post-thaw was, respectively, 89.26 ⫾ 1.56, 72.71 ⫾ 2.12, 70.20 ⫾ 2.39 and 63.02 ⫾ 2.33 (P ⬍ 0.0001), with a difference between all groups except cell viability at 30 and 120 min post-thaw (Figure 1B). Cell aliquots of 2–5 ⫻ 105 were plated in 75-cm2 culture bottles for functional assay and cell expansion evaluation post-thaw. Cells from all 15 samples adhered to the culture bottles. Cell expansion between the first and second passages post-thaw was 4.18 ⫾ 0.17 ⫻, with a doubling time of 38.06 ⫾ 1.69 h. Fourteen samples were stored in a blood bank refrigerator, in transference bags (2.5% DMSO), for 73 (36–97) h, when 500 μL were collected and plated in 25-cm2 culture bottles. Cells from all samples adhered to the bottles and expanded, preserving their morphologic characteristics (Figure 2). The UC MSC surface marker profiles (n ⫽ 10) from samples analyzed immediately after thawing and post-cultivation were similar. The cells were negative for endothelial, hematopoietic and HLA-DR surface markers and positive for mesenchymal (CD90, CD73, CD105, CD166 and CD146), adhesion molecule (CD44, CD49e, CD29, CD13 and CD54) and HLA-ABC surface markers. There was a difference between only two markers, CD73 (P ⬍ 0.002, Wilcoxon matched-pairs signed-ranks test) and CD146 (P ⬍ 0.0488) (Table I). Pre-cryopreservation results can be seen in Supplementary Table I (online only, available at http://www.informahealthcare.com/doi/ abs/10.3109/14653249.2012.677820). The samples thawed in the seventh passage (n ⫽ 5) where tested for their capacity to inhibit PBMC proliferation. The results obtained in the eight passage (the first post-thaw) were compared with the same samples tested pre-cryopreservation (third to fifth passages). The medians (ranges) of the percentage inhibition of cell proliferation pre-cryopreservation and post-thaw were, at a concentration of 1:2 (PBMC:MSC), 85.1 (70.69–92.78) and 89.31 (70.66–93.55) (P ⫽ 0.6250, Wilcoxon matched-pairs signed-rank test), at a concentration of 1:5, 72.16 (50.27–83.57) and 76.81 (40.65–86.57) (P ⫽ 0.4375), and at a concentration of 1:10, 54.74 (42.89–78.86) and 68.4 (32.00–78.10) (P ⫽ 0.4375) (Figure 3).
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K. de Lima Prata et al. Table I. Mesenchymal cell immunophenotype.a Marker CD14 CD45 CD34 CD31 HLA-DR CD338 CD90 CD731 CD105 CD1461 CD166 CD44 HLA-ABC CD49e CD29 CD13 CD54
Post-thaw
Post-cultivation
0.05 0.03 0.27 0.23 0.10 0.67 99.03 89.44 95.34 77.79 90.19 95.76 88.05 96.75 98.98 97.72 93.36
0.03 0.08 0.93 0.63 0.25 1.35 98.02 94.68 96.42 86.95 93.56 95.52 86.46 97.00 99.32 98.96 91.39
samples, except for CD338 (n ⫽ 9), were evaluated for each marker, immediately post-thaw and after cultivation. Values represent the median of the percentage of cells positive for the expression of each marker gated according to the appropriate isotype control. 1Samples with P ⬍ 0.05 (Wilcoxon matched-pairs signed-rank test). aTen
Figure 2. Post-thaw morphologic characterization of UC MSC. (A–C) UC MSC plated onto 25-cm2 culture bottles 72 h after thawing in the sixth passage. (A) 40 ⫻, (B) 100 ⫻, (C) 200 ⫻.
The UC MSC from all samples tested (n ⫽ 3) were able to differentiate into adipocytes and osteocytes (Figure 4) and showed a normal karyotype by cytogenetic analysis (G-banding of samples) (Table II). Discussion We have described a cryopreservation method for UC MSC in bags in xenofree conditions. Furthermore, we have shown that UC MSC are relatively resistant to DMSO exposure after thawing, a common event in the context of cell therapy.
We employed the BioArchive® system to cryopreserve the UC MSC because it is a computerized device that was developed to prevent exposure of the frozen cord blood units to changes in the temperature gradient. Moreover, it allows automated freezing and individually controlled storage of each unit, which has improved recovery and viability of hematopoietic stem cells after thawing (15). Cell recovery after thawing was excellent (114%), which means that virtually no cells were lost during cryopreservation, storage and the thaw/ dilution procedures. The mean recovery of viable cells 30 min after thawing was 73%. This result is better than those obtained by others (63%) (16). In addition, the method we used (PI ⫹ Annexin V, flow cytometry) to evaluate cell viability is more accurate than the most frequently used trypan blue (16–18). As expected, there was a progressive decrease of cell viability after thawing. This fact suggests that UC MSC are damaged by DMSO exposure and supports our decision to infuse the cells as soon as possible, preferably within the first 2 h after thawing. It is intuitive to assume a certain degree of cell loss (whichever cell) when exposed to DMSO; however, regarding UC MSC, we have shown the magnitude of this phenomenon. Besides cell viability, other parameters are important for evaluating UC MSC integrity, such as their immunophenotypic profile and function. We found that markers CD73 and CD146 had a decreased expression just after thawing, but
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Figure 3. UC MSC inhibition of PBMC proliferation precryopreservation and post-thaw. PBMC were polyclonally stimulated with PHA during co-cultivation with different concentrations of five UC MSC samples pre-cryopreservation (third to fifth passages) and post-thaw and cultivation (eight passage). There were no differences between the groups (P ⬎ 0.05, Wilcoxon matched-pairs test). The graphic is plotted with median and range values. The ratios are of PBMC:UC MSC.
recovered their original expression pattern when the cells were again put to cultivation. This finding suggests that there is a transitory decrease of these markers with the cryopreservation–thawing–dilution process. Probably this loss of expression, besides being transitory, does not interfere with the cell function, as the cells adhered to the culture bottle. The loss of expression of markers secondary to the cryopreservation–thawing process is not surprising. Sarugaser et al. (16) have shown that the HLA-ABC expression was stable only until the fifth passage and then lost with the cryopreservation– thawing process. However, this specific finding was not confirmed in our study, where we have shown a stronger expression of this marker in samples evaluated between the fifth and seventh passages, immediately post-thaw and after culture expansion. Most importantly, UC MSC preserved their immunomodulatory properties, despite having been submitted to two different situations that could affected their potency: the cryopreservation–thawing process, and the discrepancy in the passage (third to fifth pre-cryopreservation versus eighth post-thaw and cultivation). Also, the cryopreservation–thawing process did not alter the UC MSC differentiation
Figure 4. UC MSC differentiated into adipocytes and osteocytes post-cryopreservation. Example of a UC MSC sample differentiated into adipocytes (A; Sudan II scarlet and Harris hematoxylin staining; 630 ⫻) and osteocytes (B; von Kossa and Harris hematoxylin staining; 630 ⫻) on the 28th day of culture.
ability, or chromosome integrity, as observed by cytogenetic analysis. In summary, we think that the method described here is efficient for UC MSC cryopreservation, especially the employment of plastic bags, which facilitates their clinical use. Moreover, UC MSC are relatively resistant to DMSO exposure, but there is some decrease in viability with progressive exposure. For this reason we recommend cell infusion as soon as possible, preferably within the first 2 h of thawing.
Table II. Cytogenetic analysis of mesenchymal cells after cryopreservation. Sample UC MSC-06 UC MSC-07 UC MSC-08 aOne-hundred
Passage
Karyotype (G-band)
Structural aberrationsa
8 8 8
46, XY (50) 46, XY (50) 46, XY (50)
Not observed Not observed Not observed
conventionally stained metaphases were screened for chromatidic and chromosome gaps, and breaks, fragments, double minutes and other aberrations such as rings and dicentrics.
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Acknowledgements The authors thank Karina S. Candido, Taisa R. Fernandes, Samia R. Caruso and Camila C. O. M. Bonaldo for the laboratory assistance, and Maria Fernanda Capeli for the umbilical cord collection. They also would like to thank to CEI (Comércio Exportação e Importação de materiais médicos LTDA) for the cryopreservation bag donation. Disclosure of interests: The authors declare no conflict of interest. References 1. Horwitz EM, Le Blanc K, Dominici M, Mueller I, SlaperCortenbach I, Marini FC, et al. Clarification of the nomenclature for MSC: The International Society for Cellular Therapy position statement. Cytotherapy. 2005;7:393–5. 2. Friedenstein AJ, Piatetzky S II, Petrakova KV. Osteogenesis in transplants of bone marrow cells. J Embryol Exp Morphol. 1966;16:381–90. 3. Covas DT, Panepucci RA, Fontes AM, Silva WA Jr, Orellana MD, Freitas MC, et al. Multipotent mesenchymal stromal cells obtained from diverse human tissues share functional properties and gene-expression profile with CD146 ⫹ perivascular cells and fibroblasts. Exp Hematol. 2008;36:642–54. 4. Ringden O, Uzunel M, Sundberg B, Lonnies L, Nava S, Gustafsson J, et al. Tissue repair using allogeneic mesenchymal stem cells for hemorrhagic cystitis, pneumomediastinum and perforated colon. Leukemia. 2007;21:2271–6. 5. Le Blanc K, Samuelsson H, Gustafsson B, Remberger M, Sundberg B, Arvidson J, et al. Transplantation of mesenchymal stem cells to enhance engraftment of hematopoietic stem cells. Leukemia. 2007;21:1733–8. 6. Le Blanc K, Frassoni F, Ball L, Locatelli F, Roelofs H, Lewis I, et al. Mesenchymal stem cells for treatment of steroid-resistant, severe, acute graft-versus-host disease: a phase II study. Lancet. 2008;371:1579–86.
Supplementary material available online Supplementary Figure 1 and Table I.
7. Prockop DJ. Marrow stromal cells as stem cells for nonhematopoietic tissues. Science. 1997;276:71–4. 8. Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini F, Krause D, et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy. 2006;8:315–7. 9. Nauta AJ, Fibbe WE. Immunomodulatory properties of mesenchymal stromal cells. Blood. 2007;110:3499–506. 10. Pytlik R, Stehlik D, Soukup T, Kalbacova M, Rypacek F, Trc T, et al. The cultivation of human multipotent mesenchymal stromal cells in clinical grade medium for bone tissue engineering. Biomaterials. 2009;30:3415–27. 11. Silva WA Jr, Covas DT, Panepucci RA, Proto-Siqueira R, Siufi JL, Zanette DL, et al. The profile of gene expression of human marrow mesenchymal stem cells. Stem Cells. 2003;21: 661–9. 12. Stenderup K, Justesen J, Clausen C, Kassem M. Aging is associated with decreased maximal life span and accelerated senescence of bone marrow stromal cells. Bone. 2003;33: 919–26. 13. Covas DT, Siufi JL, Silva AR, Orellana MD. Isolation and culture of umbilical vein mesenchymal stem cells. Braz J Med Biol Res. 2003;36:1179–83. 14. Shaffer LG, Tommerup N. ISCN 2005: An International System for Human Cytogenetic Nomenclature. Basel: S. Karger; 2005. 15. Rubinstein P. Cord blood banking for clinical transplantation. Bone Marrow Transplant. 2009;44:635–42. 16. Sarugaser R, Lickorish D, Baksh D, Hosseini MM, Davies JE. Human umbilical cord perivascular (HUCPV) cells: a source of mesenchymal progenitors. Stem Cells. 2005;23: 220–9. 17. Samuelsson H, Ringden O, Lonnies H, Le Blanc K. Optimizing in vitro conditions for immunomodulation and expansion of mesenchymal stromal cells. Cytotherapy. 2009; 11:129–36. 18. Chin SP, Poey AC, Wong CY, Chang SK, Teh W, Mohr TJ, et al. Cryopreserved mesenchymal stromal cell treatment is safe and feasible for severe dilated ischemic cardiomyopathy. Cytotherapy. 2010;12:31–7.