Amniotic fluid (AF) was described as a potential source of mesenchymal stem cells (MSCs) for biomedicine purposes. Therefore, evaluation of alternative cryoprotectants and freezing protocols capable to maintain the viability and stemness of these cells after cooling is still needed. AF stem cells (AFSCs) were tested for different freezing methods and cryoprotectants. Cell viability, gene expression, surface markers, and plasticity were evaluated after thawing. AFSCs expressed undifferentiated genes Oct4 and Nanog; presented typical markers (CD29, CD44, CD90, and CD105) and were able to differentiate into mesenchymal lineages. All tested cryoprotectants preserved the features of AFSCs however, variations in cell viability were observed. In this concern, dimethyl sulfoxide (Me2SO) showed the best results. The freezing protocols tested did not promote significant changes in the AFSCs viability. Time programmed and nonprogrammed freezing methods could be used for successful AFSCs cryopreservation for 6 months. Although tested cryoprotectants maintained undifferentiated gene expression, typical markers, and plasticity of AFSCs, only Me2SO and glycerol presented workable viability ratios.
Knowledge about cryopreservation protocols that keep unchanged original stem cells properties is extremely important for cell culture and stem cells storage. For prolonged storage, cells are usually slowly frozen and stored at −196°C in liquid nitrogen. Slowly cooling avoids intracellular ice buildup, which can cause rupture of the cell membrane. Nevertheless, it can result in dehydration of the cells by formation of extracellular ice. To prevent this, an ideal cooling rate should be chosen and a cryoprotectant added. The general properties required for the cryoprotectant compounds are that they have low molecular weight, are nontoxic, and have low costs [
Cryoprotectants are divided into two main classes: intracellular agents, which penetrate inside the cells preventing ice crystals formation and membrane rupture (i.e., dimethyl sulfoxide (Me2SO), glycerol, and ethylene glycol) and extracellular compounds that do not penetrate in cell membrane and act by reducing the hyperosmotic effect present in freezing procedure. Among them are sucrose, trehalose, dextrose, and polyvinylpyrrolidone [
The main commonly used cryoprotectant, dimethylsulfoxide (Me2SO), provides a high rate of postfreezing cell survival, but several groups show that it can promote stem-cell differentiation in neuronal lineage and also presents cytotoxicity at room temperature [
MSCs are multipotent cells capable to differentiate into several lineages
AF obtained in second trimester of pregnancy is composed of a wide variety of cells derived mainly from fetal exfoliation during development. Among these cells, some have adherent properties and express undifferentiated markers such as Oct-4 gene. These cells are also capable of self-renewal and differentiation into mesodermal tissues in culture. They have been classified as amniotic fluid mesenchymal stem cells (AFSCs) [
Here we evaluated the effects of two cryopreservation methods (time-programmed and nonprogrammed temperature freezing) and four distinct cryoprotectants in the AFSCs viability and main characteristics maintenance after liquid nitrogen storage.
All culture media and chemical reagents were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Fetal bovine serum and antibiotics were purchased from Invitrogen (Carlsbad, CA, USA). Primers were from IDT (Integrated DNA Technologies, San Diego, CA, USA). Ntera-2.c1D1 (human pluripotent testicular carcinoma/ATCC CRL-1973) was purchased from American Cell Type Collection (Manassas, VA, USA).
Human amniotic fluid samples were collected during scheduled amniocentesis at 12 to 18 weeks of gestation for diagnosis reasons. Collection procedure was performed under aseptic conditions using a 22-gauge needle and under ultrasonographic control. All samples were obtained after signed informed consent. The protocol was approved by the Ethics Committee from University of Sao Paulo School of Medicine, Brazil (no.: 0410-09).
After collection, each sample was centrifuged at 450 × g for 10 min at room temperature. The resulting pellets were washed with PBS (phosphate buffer saline), suspended in minimal essential medium—
For growth curve, AFSCs were seeded in 48-well plates (Corning, USA), and cultivated for 12 days in a platting density of 5,000 cells/cm2. Cell number was assessed daily in triplicate by trypan blue staining in hemocytometer (Optik Labor, Friedrichshofen, Germany) counting. Cell population doubling time was calculated by the following formula:
To evaluate undifferentiated state of AFSCs, specific gene expressions were determined: Oct-4 and Nanog. Total RNA was extracted using Trizol Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Purity of the RNA was assessed by determining the ratio of absorbance at 260 nm to that at 280 nm. The reactions were performed with the kit Illustra Ready To Go (GE Healthcare, Buckinghamshire, UK) in a PTC-200 thermal cycler (MJ Research Inc., Watertown, USA). Approximately 100 ng of total RNA were required for reactions.
RT-PCR was performed initially with a step of 50°C for 30 min for reverse transcription of RNA and denaturation at 95°C for 5 min. This was followed by 35 cycles (94°C for 30 sec, 57°C for 30 sec, and 72°C for 60 sec; final extension at 72°C for 8 min). All used sense and antisense primers are described in Table
RT-PCR primer sequences.
Gene | Primers | PCR products (bp) | Tm (°C) |
---|---|---|---|
Oct-4 | 5′- cgt gaa gct gga gaa gga gaa gct g -3′ | 247 | 61 |
Nanog | 5′-cat gag tgt gga tcc agc ttg-3′ | 191 | 52 |
Osteocalcin | 5′-atg aga gcc ctc aca ctc ct-3′ | 359 | 55 |
Osteopontin | 5′-cat ggc atc acc tgt gcc ata cc-3′ | 331 | 57 |
Wnt-1 | 5′-cac gac act cgt gca gta cgc-3′ | 233 | 58 |
Collagen 2A1 | 5′-cca gga cca aag gga cag aaa g-3′ | 398 | 57 |
Perlecan | 5′-cat aga gac cgt cac agc aag-3′ | 300 | 55 |
Syndecan | 5′-cct tca cac tcc cca cac-3′ | 410 | 54 |
PPAR- | 5′-gct gtt atg ggt gaa act ctg-3′ | 351 | 55 |
Lipoprotein lipase | 5′-cag caa aac ctt ca tggt-3′ | 72 | 53 |
5′-tgg cac cac acc ttc tac aat gag c-3′ | 396 | 60 |
Specific surface proteins of cultured AFSCs were evaluated by flow cytometry (FacsCalibur cytometer, Becton Dickinson) and analyzed in CellQuest software.
To induce osteogenic differentiation, AFSCs at 70–80% confluence were cultured in osteogenic medium (
To induce adipose tissue differentiation, AFSCs were treated with adipogenic medium:
To induce chondrogenic differentiation, AFSCs were grown in
AFSCs (third passage) were frozen in the final concentration of 106 cells/mL in freezing medium (
Data are expressed as mean ± SEM. One-way or two-way analysis of variance (ANOVA) was applied to the means to determine statistical differences between experimental groups followed by Bonferroni or Dunnett’s tests. Differences between mean values were considered significant when
AFSCs were isolated of 35 of 41 samples by their adherent properties in plastic surface and appeared as colonies in the culture flask. They consisted of fibroblast-like cell morphology. Doubling time for AFSCs calculated after growth curve was 30 ± 4 h (Figure
AFSCs growth curve and doubling time. AFSCs (5,000 cells/cm2) were seeded and cultivated in 46-well plates for 12 days. At the end of each day, cells were washed, trypsinizated, and counted in hemocytometer. Each point refers to triplicate from two different AF samples. Cell doubling time calculated was 30 hours (±4 h).
Cultured AFSCs expressed the transcription factors Oct-4 at different passages, as determined by RT-PCR. Oct-4 is a key marker expressed in embryonic stem cells and cancer cells and is responsible for the maintenance of the undifferentiated state. The homeodomain gene Nanog, present in pluripotent human cells, was also expressed in cultured AFSCs confirming the undifferentiated state (Figure
Undifferentiated gene expression (Oct-4 and Nanog). AFSCs total RNA was extracted from 3 different samples by Trizol method, gene expression evaluated by RT-PCR, and electrophoresed and visualized under ultraviolet light in 2% agarose gel stained with ethidium bromide.
The results obtained by flow cytometry demonstrated that stem cells isolated from amniotic fluid have a strong positivity for mesenchymal markers, such as CD29 (
AFSCs membrane proteins were evaluated by flow cytometry with CellQuest software. Acquisitions were shown in histograms. AFSCs were positive for CD29 (98.27 ± 1.23%), CD44 (98.28 ± 1.65%), CD90 (97.69 ± 1.84%), CD105 (98.77 ± 0.64%) and were negative for CD14 (1.78 ± 1.06%), CD34 (1.70 ± 1.14%), and CD45 (1.80 ± 0.74%). IgG1-FITC and IgG1-PE antibodies were utilized as isotype controls. The number of acquired events was 20,000 in each acquisition.
AFSCs were cultivated in specific differentiation medium, as described, to evaluate their plasticity and multipotency. Osteogenic differentiation was confirmed after 21 days by visualization of the calcium deposits seen with Alizarin Red and von Kossa staining (Figure
AFSCs plasticity. AFSCs were seeded in 6-well plates and cultivated with osteogenic, adipogenic, and chondrogenic inductor medium for 3 weeks. (a) Control cells cultivated without inductor medium (magnification: 40x). (b) Osteogenic differentiation was assessed by von Kossa (calcium deposits in black) and (c) Alizarin Red (calcium deposits in red) staining. Adipogenic differentiation was assessed by Oil Red O staining. (d) Differentiated cells without staining (magnification: 40x), (e) differentiated cells stained by Oil Red O (cytoplasmic lipids droplets in red, 400x). (f) Chondrogenic differentiation was confirmed with H&E (magnification: 400x) (g) and immunofluorescence staining with antibodies against collagen type II (400x). (h) Osteogenic gene expression was determined by RT-PCR; products were electrophorezed and visualized under ultraviolet light in 2% agarose gel stained with ethidium bromide (100 bp molecular weight, control (water), osteopontin [OPN], osteocalcin [OCN] and Wnt-1). (i) Typical adipogenic gene expression was determined by RT-PCR (100 bp molecular weight, control (water), PPAr-
Adipogenic differentiation was confirmed by Oil Red O staining. Cytoplasmic lipid droplets appeared stained in red. They also expressed adipocytes genes as PPAr-
AFSCs were also able to differentiate into chondrogenic tissue after stimulation with TGF-
AFSCs viability after thawing did not present statistical difference when compared the nonprogrammed and programmed time freezing protocols after 3 and 6 months storage (Figure
AFSCs viability comparing nonprogrammed and programmed freezing protocols. we did not have statistic differences in AFSCs viability when we compared the two freezing methods tested. (a) AFSCs viability after 6 months in programmed and nonprogrammed freezing using different cryoprotectants. (b) AFSCs viability after 3 months in programmed and nonprogrammed freezing using different cryoprotectants. M-Me2SO, G-glycerol, S-sucroses and T-trehalose. Means were compared by one-way ANOVA Dunnett posttest (*
All evaluated cryoprotectants preserved the basic features of AFSCs, as Oct-4 and Nanog genes expression (Figure
After nonprogrammed freezing, AFSCs (
Nonprogrammed | CD14 PE | CD34 FITC | CD45 FITC | CD29 PE | CD44 FITC | CD105 PE |
---|---|---|---|---|---|---|
Me2SO 5% | 1.22 ± 0.25 | 0.29 ± 0.25 | 0.35 ± 0.16 | 98.30 ± 1.05 | 99.26 ± 0.39 | 99.01 ± 1.03 |
Me2SO 10% | 1.02 ± 0.18 | 0.74 ± 0.22 | 5.80 ± 2.02 | 99.81 ± 0.11 | 99.90 ± 0.07 | 99.26 ± 0.36 |
Glycerol 5% | 0.94 ± 0.44 | 0.44 ± 0.16 | 0.84 ± 0.31 | 99.54 ± 0.26 | 99.91 ± 0.06 | 98.92 ± 0.95 |
Glycerol 10% | 0.87 ± 0.56 | 0.09 ± 0.27 | 0.16 ± 0.09 | 99.10 ± 0.33 | 99.09 ± 0.46 | 99.33 ± 0.51 |
Sucrose 30 mM | 1.06 ± 0.23 | 0.81 ± 0.51 | 1.08 ± 0.73 | 99.74 ± 0.04 | 99.69 ± 0.18 | 99.44 ± 0.12 |
Sucrose 60 mM | 0.95 ± 0.65 | 1.14 ± 0.89 | 3.49 ± 0.88 | 99.43 ± 0.25 | 99.57 ± 0.32 | 98. 26 ± 1.39 |
Trehalose 60 mM | 1.93 ± 0.26 | 0.36 ± 0.31 | 0.37 ± 0.26 | 99.86 ± 0.28 | 99.98 ± 0.01 | 98.24 ± 1.17 |
Trehalose 100 mM | 0.82 ± 0.72 | 0.48 ± 0.28 | 0.79 ± 0.17 | 99.91 ± 0.05 | 99.08 ± 0.74 | 99.54 ± 0.09 |
After programmed freezing, AFSCs (
Programmed | CD14 PE | CD34 FITC | CD45 FITC | CD29 PE | CD44 FITC | CD105 PE |
---|---|---|---|---|---|---|
Me2SO 5% | 1.88 ± 0.35 | 0.58 ± 0.22 | 0.85 ± 0.53 | 96.06 ± 2.61 | 97.03 ± 2.06 | 98.82 ± 1.06 |
Me2SO 10% | 0.92 ± 0.46 | 0.75 ± 0.13 | 0.76 ± 0.44 | 99.67 ± 0.16 | 99.88 ± 0.10 | 99.02 ± 0.15 |
Glycerol 5% | 0.91 ± 0.74 | 0.18 ± 0.08 | 0.39 ± 0.29 | 99.48 ± 0.40 | 99.85 ± 0.16 | 99.51 ± 0.17 |
Glycerol 10% | 1.76 ± 0.96 | 1.07 ± 0.97 | 1.12 ± 1.01 | 99.71 ± 0.16 | 99.87 ± 0.08 | 99.11 ± 0.39 |
Sucrose 30 mM | 0.62 ± 0.48 | 0.22 ± 0.14 | 0.48 ± 0.15 | 99.29 ± 0.19 | 99.95 ± 0.03 | 99.65 ± 0.06 |
Sucrose 60 mM | 1.36 ± 0.08 | 0.96 ± 0.06 | 1.51 ± 0.83 | 99.35 ± 0.28 | 99.17 ± 0.66 | 99.02 ± 0.20 |
Trehalose 60 mM | 1.25 ± 0.41 | 0.31 ± 0.25 | 0.48 ± 0.25 | 97.77 ± 1.31 | 99.85 ± 0.11 | 98.90 ± 0.66 |
Trehalose 100 mM | 0.53 ± 0.88 | 0.46 ± 0.36 | 0.60 ± 0.56 | 99.50 ± 0.36 | 99.83 ± 0.18 | 98.94 ± 0.76 |
AFSCs were thawed, total RNA was extracted, and Oct-4 and Nanog gene expression was determined by RT-PCR in 2% agarose gel stained with ethidium bromide after freezing with different cryoprotectants. Human
After thawing, AFSCs freezed with different cryoprotectants ((b) Me2SO, (c) glycerol, (d) sucrose, and (e) trehalose) were cultivated with osteogenic and adipogenic inductor medium. Differentiation was confirmed by Alizarin Red and Oil Red O staining after 21 days. (a) Control cells without inductor. Calcium deposits appear in red. All cryoprotectants tested maintained osteogenic and adipogenic differentiation potential of AFSCs after storage for 6 months.
AFSCs viability comparing different cryoprotectants (Me2SO, glycerol, sucrose, and trehalose) and programmed and nonprogrammed freezing protocols after 3 and 6 months. (a) viability in nonprogrammed method for 3 months; (b) viability in programmed method for 3 months; (c) viability in nonprogrammed method for 6 months; (d) viability in programmed method for 6 months. 10% Me2SO showed the higher viability rate similar to NR control in all cases. NR-AFSCs viability control measured before freezing procedure. M-Me2SO, G-glycerol, S-sucrose, and T-trehalose. Means were compared by one-way ANOVA Dunnett’s (***
The interest of mesenchymal stem cells in the scientific community has arisen due to their peculiar characteristics. Among them, the highlights are proliferative capacity and differentiation potential [
Primary culture of human amniotic fluid revealed the presence of fibroblast-like cells capable of adhesion and colonies formation in plastic dish surface. These cells were classified as mesenchymal stem cells by their characteristics, as follows.
AFSCs doubling time (30 hours) founded was similar with that described elsewhere [
Key gene expression profile, such as Oct-4 and Nanog, is essential to determine the undifferentiated stem cells status. These genes are commonly found in pluripotent stem cells and cancer cells [
Cell cultures require cooling methods for long-time storage, and the choice of freezing protocol used must be done carefully to avoid cell death and, mainly, to maintain the cell characteristics, as in this case, AFSCs proliferation and plasticity properties [
The cooling rate is a critical step during freezing. If cooling is sufficiently slow, the cell is able to lose water rapidly enough by exosmosis to concentrate the intracellular solutes sufficiently to eliminate supercooling and maintain the chemical potential of intracellular water in equilibrium with that of extracellular water. The result is that the cell dehydrates and does not freeze intracellularly. But if the cell is cooled too rapidly, it is not able to lose water fast enough to maintain equilibrium; it becomes increasingly supercooled and eventually attains equilibrium by freezing intracellularly. Intracellular ice formation leads to cell death [
It was observed that the two methods had no effect when compared with each other in cellular viability after thawing. Therefore, a nonprogrammed method should make cryopreservation more convenient and less costly because the use of complex equipment for controlled-rate freezing is avoided.
Cryoprotectants are essential to maintain the cell viability and function for storage at very low temperatures, including for amniotic fluid-derived stem cells [
Glycerol, another intracellular cryoprotectant, was compared with Me2SO. Glycerol is also widely used in concentrations of 5 and 10%, in cell cultures. It is described that it does not promote cellular toxicity [
Disaccharides such as sucrose and trehalose have also been widely used as natural cryoprotectants, as excipients for freeze drying and as stabilizers during dehydration. The precise mechanism by which disaccharides act to preserve biological systems during freezing and drying is not well understood. The fact that they do not enter in cells is the main advantage, facilitating their removal after thawing [
All tested cryoprotectants maintained the Oct-4 and Nanog gene expression and did not alter membrane markers or the plasticity of AFSCs post thawing, which were able to differentiate into bone and fat tissues after thawing.
Human amniotic fluid collected in the second trimester of gestation contains mesenchymal stem cells which show a high rate of proliferation
The authors declare no conflict of interests.
This paper was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Financiadora de Estudos e Projetos (FINEP), and Instituto Nacional de Ciência e Tecnologia-Fluidos Complexos (INCT-FCx), Brazil.