Cryopreservation does not alter karyotype, multipotency, or NANOG/SOX2 gene expression of amniotic fluid mesenchymal stem cells

Cryopreservation of mesenchymal stem cells from amniotic fluid is of clinical importance, as these cells can be harvested during the prenatal period and stored for use in treatments. We examined the behavior of mesenchymal stem cells from human amniotic fluid in culture that had been subjected to cryopreservation. We assessed chromosomal stability through karyotype analysis, determined whether multipotent capacity (differentiation into adipogenic, chondrogenic, and osteogenic cells) is maintained, and analyzed SOX2 and NANOG expression after thawing. Five amniotic fluid samples were cryopreserved for 150 days. No chromosomal aberrations were observed. The expression levels of NANOG and SOX2 also were quite similar before and after cryopreservation. Capacity for differentiation into adipogenic, chondrogenic, and osteogenic tissues also remained the same. We conclude that cryopreservation of amniotic fluid does not alter karyotype, NANOG/SOX2 gene expression, or multipotent capacity of stem cells that have been collected from amniotic fluid during pregnancy.     

Human amniotic epithelial cells induce apoptosis of cancer cells: a new anti-tumor therapeutic strategy

Background aimsAmniotic membrane (AM), the innermost layer of human placenta, is composed of a single layer of epithelial cells, a basement membrane and an avascular stroma. The AM has many functions and properties, among which angiogenic modulatory and immunoregulatory effects are applicable in cancer therapy. Because these functions belong to amniotic epithelial cells, in this study we compared the anti-cancer effect of amniotic epithelial cells and the whole AM.MethodsThe effect of the AM and the amniotic epithelial cells on cancer cell apoptosis was evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium assay, terminal deoxynucleotidyl transferase dUTP nick end labeling assay and immunocytochemistry. The effect of the AM on angiogenesis in conditions both with and without epithelial cells was also evaluated using rat aortic ring assay.ResultsThere was a decrease in cancer cell viability after adding either AM or amniotic epithelial cell supernatant to cancer cells. A significant increase in caspase-3 and caspase-8 expression in cancer cells treated with amniotic epithelial cell supernatant was observed. The recorded media also demonstrated the possible induction of apoptosis in cancer cells treated with the amniotic epithelial cell supernatant. In the aorta ring assay, the AM showed an anti-angiogenic effect in the presence of its epithelial cells; however, this effect was altered to initiate angiogenesis when amniotic epithelial cells were removed from the AM.ConclusionsThese results suggest that amniotic epithelial cells, with their anti-angiogenic effect and induction of apoptosis, are candidates for cancer therapeutic agents in the near future.     

Serum-free cryopreservation of human amniotic epithelial cells before and after isolation from their natural scaffold

Amniotic epithelial cells are a promising source for stem cell-based therapy through their potential capacity to differentiate into the cell lineages of all three germ layers. Long-term preservation is necessary to have a ready-to-use source of stem cells, when required. Reduced differentiation capability, decrease of viability and use of fetal bovine serum (FBS) are three drawbacks of clinical application of cryopreserved stem cells. In this study, we used human amniotic fluid instead of animal serum, and evaluated viability and multipotency of amniotic epithelial cells after cryopreservation in suspension and compared with those cryopreserved on their natural scaffold (in situ cryopreservation). There was no significant difference in viability of the cells cryopreserved in amniotic fluid and FBS. Also, the same results were achieved for expression of pluripotency marker OCT-4 when FBS was replaced by amniotic fluid in the samples with the same cryoprotectant. The cells cryopreserved in presence of scaffold had a higher level of viability compared to the cells cryopreserved in suspension. Although, the number of the cells expressed OCT-4 significantly decreased within cryopreservation in suspension, no decrease in expression of OCT-4 was observed when the cells cryopreserved with their natural scaffold. Upon culturing of post-thawed cells in specific lineage differentiating mediums, the markers of neuronal, hepatic, cardiomyocytic and pancreatic were found in differentiated cells. These results show that replacement of FBS by amniotic fluid and in situ cryopreservation of amniotic epithelial cells is an effective approach to overcome limitations related to long-term preservation including differentiation during cryopreservation and decrease of viability.     

Augmented Dried versus Cryopreserved Amniotic Membrane as an Ocular Surface Dressing

Purpose

Dried amniotic membrane (AM) can be a useful therapeutic adjunct in ophthalmic surgery and possesses logistical advantages over cryopreserved AM. Differences in preservation techniques can significantly influence the biochemical composition and physical properties of AM, potentially affecting clinical efficacy. This study was established to investigate the biochemical and structural effects of drying AM in the absence and presence of saccharide lyoprotectants and its biocompatibility compared to cryopreserved material.

Methods

AM was cryopreserved or dried with and without pre-treatment with trehalose or raffinose and the antioxidant epigallocatechin (EGCG). Structural and visual comparisons were assessed using electron microscopy. Localisation, expression and release of AM biological factors were determined using immunoassays and immunofluorescence. The biocompatibility of the AM preparations co-cultured with corneal epithelial cell (CEC) or keratocyte monolayers were assessed using cell proliferation, cytotoxicity, apoptosis and migration assays.

Results

Drying devitalised AM epithelium, but less than cryopreservation and cellular damage was reduced in dried AM pre-treated with trehalose or raffinose. Dried AM alone, and with trehalose or raffinose showed greater factor retention efficiencies and bioavailability compared to cryopreserved AM and demonstrated a more sustained biochemical factor time release in vitro. Cellular health assays showed that dried AM with trehalose or raffinose are compatible and superior substrates compared to cryopreserved AM for primary CEC expansion, with increased proliferation and reduced LDH and caspase-3 levels. This concept was supported by improved wound healing in an immortalised human CEC line (hiCEC) co-cultured with dried and trehalose or raffinose membranes, compared to cryopreserved and fresh AM.

Conclusions

Our modified preservation process and our resultant optimised dried AM has enhanced structural properties and biochemical stability and is a superior substrate to conventional cryopreserved AM. In addition this product is stable and easily transportable allowing it to be globally wide reaching for use in clinical and military sectors.     

Cryopreservation of brain tissue for primary culture.

Factors affecting recovery of brain cells from cryopreserved cerebral tissues of fetal rats were examined based on yields of viable cells on cell culture. Favorable preservation was obtained with freezing small pieces (less than 1 mm cube) of brain tissues rather than whole tissues or dissociated single cells, and use of 10% dimethylsulfoxide as a cryoprotectant in liquid nitrogen. As for cell preparation procedures, cell survival was improved when tissues were heated at 32 degrees C during papain digestion and centrifugation. Under favorable conditions, the number of brain cells recovered from cryopreserved tissues corresponded to 20-30% of those from fresh control tissues. Immunocytochemical characteristics of cultured neurons, astrocytes, and oligodendrocytes from cryopreserved and fresh tissues were indistinguishable. Semi-quantitive analyses of microtubule-associated protein-2 (MAP-2) and synaptophysin revealed that there was no difference in the amounts of these markers between cultures from both fresh and cryopreserved tissues. These results suggest that most of all cell types including neurons were equally susceptible to the cryopreservation procedures. We concluded that cryopreservation in liquid nitrogen is an effective method for preservation of embryonic brain tissues for later use in cell culture studies.     

Viability of pulp stromal cells in cryopreserved deciduous teeth

The cryopreservation of exfoliated deciduous teeth and harvesting of stem cells from them as required would reduce the costs and efforts associated with banking stem cells from primary teeth. The aim of this study was determine whether the viability of pulp stromal cells from deciduous teeth was influenced by the cryopreservation process itself or the period of cryopreservation. In total, 126 deciduous teeth were divided into three groups: (1) fresh, (2) cryopreserved for <3 months (cryo<3), and (3) cryopreserved for 3–9 months (cryo3–9). The viability of the pulp tissues was compared among the three groups by evaluating the outgrowth from pulp tissues and cell activity within those pulp tissues. In addition, the terminal deoxynucleotidyl transferase-mediated dUTP–biotin nick end labeling (TUNEL) assay was performed to compare cell apoptosis within fresh pulp tissue and pulp tissue that had been cryopreserved for 4 months. The outgrowth from and cell activity within the pulp tissues did not differ significantly between the fresh and cryo<3 pulp tissues. However, these parameters were significantly reduced in the cryo3–9 pulp tissue. In TUNEL assay, 4-month cryopreserved pulp tissues has more apoptotic cells than fresh group. In conclusion, it is possible to acquire pulp stromal cells from cryopreserved deciduous teeth. However, as the period of cryopreservation becomes longer, it is difficult to get pulp cells due to reduced cell viability.     

The effects of preservation procedures on antibacterial property of amniotic membrane

Amniotic membrane (AM), the innermost layer of the fetal membranes, has been widely employed in the surgical reconstruction and tissue engineering. Expression of the antimicrobial peptides such as defensins, elafin and SLPI which are essential elements of the innate immune system results in antibacterial properties of the AM. Preservation is necessary to reach a ready-to-use source of the AM. However, these methods might change the properties of the AM. The aim of this study was to evaluate antibacterial properties of the AM after preservation. Antibacterial property of the fresh AM was compared with cryopreserved and freeze-dried AM by modified disk diffusion method. Staphylococcus aureus ATCC 25923, Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC 25922 and two clinical isolated strains of E. coli were cultured in Mueller Hinton agar and a piece of the AM was placed on agar surface. After 24 h incubation, the inhibition zone was measured. In addition, one of the most important antibacterial peptides, elafin, was measured by ELISA assay before and after preservations procedures. Antibacterial properties of the AM were maintained after cryopreservation and freeze-drying. However, the inhibition zone was depending on the bacterial strains. The cryopreservation and freeze-drying procedures significantly decreased elafin which shows that antibacterial property is not limited to the effects of amniotic cells and the other components such as extracellular matrix may contribute in antibacterial effects. The promising results of this study show that the preserved AM is a proper substitute of the fresh AM to be employed in clinical situations.     

Regrowth of embryogenic tissues of <Emphasis Type="Italic">Pinus nigra</Emphasis> following cryopreservation

Embryogenic tissues of a conifer species Pinus nigra Arn. have been cryopreserved by a slow-freezing method. The effect of cryoprotective compounds such as maltose, sucrose or sorbitol (each 0.5 M) combined with 7.5% (v/v) DMSO has been tested. After thawing, the following parameters have been investigated: tissue regrowth 6 weeks after thawing, and post-thaw growth evaluated as fresh mass accumulation as well as genetic stability in post-thaw period. The parameters mentioned have been compared in both cryopreserved and non-cryopreserved tissues. Out of eight cell lines used in experiments, seven survived cryopreservation (87.5% regrowth), although cell line and treatment effects were observed. In most cell lines, sucrose or maltose pretreatments were superior over sorbitol. In the regrown cell lines, the post-thaw growth as fresh mass accumulation was not negatively influenced by cryopreservation. No genetic variation was observed in cryopreserved tissues using a RAPD approach.     

Cryopreservation of composite tissues and transplantation: preliminary studies

Despite advances in cryobiology, the reliable cryopreservation of complex tissues has not yet been achieved. This study evaluates the viability of cryopreserved composite flaps and demonstrates the feasibility of their transplantation. Epigastric flaps were harvested from male Lewis rats. 1.5 M dimethyl sulfoxide (Me₂SO) was used as the initial cryoprotectant agent (CPA). Samples were frozen at controlled rate to −140 °C and transferred to liquid nitrogen for at least two weeks. Hematoxylin and eosin (H/E) staining, MTT tetrazolium salt assay, and factor VIII immunostaining were used to evaluate the overall histology, epithelial viability, and vascular endothelial integrity, respectively, of cryopreserved flaps. For the in vivo phase, flaps were isotransplanted to 35 recipient animals, divided into three groups: fresh (n = 10), perfused (n = 8), and cryopreserved (n = 17). Blood vessel patency was assessed via Doppler at 1, 7, and 60 days post-transplantation. For in vitro studies, cryopreserved samples (10/10) retained normal cell architecture and vascular endothelial integrity upon H/E and factor VIII staining. The viability index of cryopreserved composite flap skin (n = 10) was 11.17 ± 2.01, which was not significantly different from fresh controls (n = 10, 12.15 ± 1.32). All transplanted flaps in the fresh and perfusion groups survived with healthy color and hair growth at 60 days after operation. Survival in the cryopreserved group ranged from 2 to 60 days, with a mean of 12 days. These results demonstrate that the long term survival of cryopreserved composite tissue transplants is possible. Further studies are needed to refine protocols for the reliable cryopreservation of composite parts.     

A comparison of lyophilized amniotic membrane with cryopreserved amniotic membrane for the reconstruction of rabbit corneal epithelium

Many researchers have employed cryopreserved amniotic membrane (CAM) in the treatment of a severely damaged cornea, using corneal epithelial cells cultured on an amniotic membrane (AM). In this study, two Teflon rings were made for culturing the cells on the LAM and CAM, and were then used to support the AM, which is referred to in this paper as an Ahn’s AM supporter. The primary corneal epithelial cells were obtained from the limbus, using an explantation method. The corneal epithelium could be reconstructed by culturing the third-passage corneal epithelial cells on the AM. A lyophilized amniotic membrane (LAM) has a higher rate of graft take, a longer shelf life, is easier to store, and safer, due to gamma irradiation, than a CAM. The corneal epithelium reconstructed on the LAM and CAM, supported by the two-Teflon rings, was similar to normal corneal epithelium. However, the advantages of the LAM over that of the CAM make the former more useful. The reconstruction model of the corneal epithelium, using AM, is considered as a goodin vitro model for transplantation of cornel epithelium into patients with a severely damaged cornea.     

The clinical outcomes of fresh versus cryopreserved CD19-directed chimeric antigen receptor T cells in non-Hodgkin lymphoma patients

CD19-directed chimeric antigen receptor T (CAR-T) cells have been widely reported in the therapy of relapsed/refractory non-Hodgkin lymphoma (NHL). Both cryopreserved and fresh formulations of CAR-T have been used in previous studies. However, quite a few studies investigated the effects of cryopreservation on the clinical outcomes of CAR-T cells. Here we retrospectively analyzed a phase I/II clinical trial of CD19-directed CAR-T cells in NHL patients, and compared the safety and efficacy of cryopreserved and fresh CAR-T products. All CAR-T cells were prepared using the same manufacturing process except the formulation step. Fifteen patients were infused with cryopreserved/thawed CAR-T cells, and 8 patients were treated with fresh CAR-T cells. Comparative overall response rates and in vivo expansion kinetics of CAR-T cells were observed between the cryopreserved cohort and fresh cohort. The occurrence rates of cytokine release syndrome and neurotoxicity were also similar in both groups. Patients in the fresh cohort showed higher incidence of acute hematological toxicity including anemia, hypoleukemia, and thrombocytopenia. This study demonstrated that cryopreservation showed negligible effects on the efficacy of CD19-directed CAR-T cells, but endowed CAR-T cells with higher safety in NHL patients, supporting the application of cryopreserved CAR-T products for NHL therapy.     

Investigation of interleukin-1β release from cryopreserved blood stimulated with endotoxin

The feasibility of an indigenously developed ELISA method to determine cytokine response to wide spectrum of pyrogenic stimuli utilizing fresh human whole blood is limited by the availability of healthy donors. The possibility of using cryopreservation of pooled human blood for detection of cytokine response to lipopolysaccharide is explored in this study. The effect of cryopreservation on blood parameters, cellular morphology and cytokine response were compared with that of the pooled fresh blood and cryopreserved blood from single and multiple donors. In vitro pyrogenic stimulation with 0.5 and 5 EU of LPS was monitored on fresh and cryopreserved pooled blood from single and multiple donors. The release of IL-1β was quantitated by Sandwich ELISA (1, 10, 25, 45 and 75 days) after storage. The results indicated that the cryopreserved blood displayed enhanced IL-1β release on stimulation with LPS, when compared to fresh blood. The maximum release of IL-1β level was observed at 2 h when 5 EU of LPS was treated with pooled fresh blood which is similar to that of fresh blood. After 75 days storage of pooled cryopreserved blood the IL-1β release was maximum at 9 and 15 h when treated with 5 and 0.5 EU of LPS. Observations of the study suggest that cryopreserved pooled blood is an economically and experimentally viable alternative to fresh blood. This investigative study promises short term storage and regular supply of non-allergic, pathogen free human blood for the detection of interleukin-1β for the evaluation of in vitro pyrogenicity.     

Safety and efficacy of human amniotic membrane in primary pterygium surgery

Grafts made from human amniotic membrane are used to prevent recurrence of pterygium after excision. The success of the procedure can be affected by the quality of preparation and preservation of the grafts. We prospectively evaluated the safety and efficacy of cryopreserved amniotic membrane prepared at the research tissue bank of the Biotechnology Research Center in Tripoli, Libya, and used as adjunct therapy in primary pterygium excision. Twenty-six patients (15 males and 11 females) aged 21–78 years and indicated for primary pterygium excision were transplanted at the Tripoli Eye Hospital with the amniotic membrane grafts. Sixteen patients (62 %) were available for all three follow-up visits scheduled at 1, 3 and 6 months post-surgery. By the third visit, two patients (12.5 %) developed granuloma and three (18.8 %) had pterygium recurrence. The grafts were used after cryopreservation for ≤180 days or >180 days, but statistical analysis showed that the complications were not associated with the length of storage. Moreover, the high rate of complications in this study was not caused by use of cryopreserved AM. In conclusion, locally produced cryopreserved AM is safe as an adjunct therapy for treatment of primary pterygium excision.     

The effects of preservation procedures on amniotic membrane's ability to serve as a substrate for cultivation of endothelial cells

Amniotic membrane (AM) has been used as a scaffold for the ex vivo expansion of different types of cells and a cell delivery matrix in regenerative medicine. Since the preservation procedures can influence the AM properties for experimental and clinical purposes, this study was established to investigate the feasibility of using the AM after different preservation methods to serve as substrates for endothelial cell expansion ex vivo. The effects of cryopreservation and lyophilization were evaluated on mechanical and histological characteristics of the AM, and the results were compared with the fresh AM. The ECM components of the basement membrane were well conserved in all groups. Although lyophilization resulted in more histological changes and lower level of physical variables including thickness, F_max, elongation at break and suture retention than the fresh and cryopreserved AM, endothelial cells grown on the lyophilized AM were better attached to the basement membrane. Cytotoxicity assay by MTT showed that the lyophilized AM is a compatible substrate for endothelial cells cultivation. The findings of this study suggest that the lyophilized AM is a suitable matrix for cultivation of endothelial cells due to this fact that lyophilization led to exposure of basement membrane of the AM.     

The effects of preservation procedures on amniotic membrane’s ability to serve as a substrate for cultivation of endothelial cells

Amniotic membrane (AM) has been used as a scaffold for the ex vivo expansion of different types of cells and a cell delivery matrix in regenerative medicine. Since the preservation procedures can influence the AM properties for experimental and clinical purposes, this study was established to investigate the feasibility of using the AM after different preservation methods to serve as substrates for endothelial cell expansion ex vivo. The effects of cryopreservation and lyophilization were evaluated on mechanical and histological characteristics of the AM, and the results were compared with the fresh AM. The ECM components of the basement membrane were well conserved in all groups. Although lyophilization resulted in more histological changes and lower level of physical variables including thickness, F_max, elongation at break and suture retention than the fresh and cryopreserved AM, endothelial cells grown on the lyophilized AM were better attached to the basement membrane. Cytotoxicity assay by MTT showed that the lyophilized AM is a compatible substrate for endothelial cells cultivation. The findings of this study suggest that the lyophilized AM is a suitable matrix for cultivation of endothelial cells due to this fact that lyophilization led to exposure of basement membrane of the AM.     

Reversal of myofibroblasts by amniotic membrane stromal extract

Myofibroblasts play an important role in morphogenesis, inflammation, and fibrosis in most tissues. The amniotic membrane stroma can maintain keratocytes in cultures and prevent them from differentiating into myofibroblasts. However, it is unknown whether the AM stroma can also reverse differentiated myofibroblasts. In this study, we found that amniotic membrane stromal cells (AMSCs), which adopted fibroblastic phenotype in vivo, quickly and completely differentiated into myofibroblasts during ex vivo culture in DMEM/FBS on plastic within 2 passages. When cultured on type I collagen, the myofibroblasts maintained their phenotype, however, when these myofibroblasts were re-seeded onto a cryopreserved amniotic membrane stromal surface, they reversed to the fibroblast phenotype. Moreover, we found that the amniotic membrane stromal extract not only helps maintain primary AMSCs fibroblastic phenotype in vitro, but also can reverse differentiated myofibroblasts back to fibroblasts. This reversal was not coupled with cell proliferation. We concluded that the amniotic membrane stroma contains soluble factors that can regulate the mesenchymal cell differentiation. Further investigation into the identity of these factors and the control mechanisms may unravel a new scar-reversing strategy.     

Cryopreservation-induced enhancement of interleukin-2 production in human peripheral blood mononuclear cells.

To better understand the effects of cryopreservation on various immunocompetent cell functions, we have examined the interleukin-2 (IL-2)-producing activities of frozen mononuclear cells (MNCs) from healthy subjects. The mechanisms responsible for the observed effects were also analyzed. Both the unfractionated and monocyte-depleted, frozen MNCs produced significantly larger quantities of IL-2 than fresh cells. Similar to freezing, L-leucine methyl ester (Leu-OMe) treatment (to eliminate IL-1 and prostaglandin E-2 (PGE-2)-secreting cells) also increased the IL-2-producing activities of fresh cells, but freezing no longer enhanced the production of IL-2 by Leu-OMe-treated cells, suggesting that (1) both the freezing process and Leu-OMe treatment have similar effects on IL-2 production, (2) the increased IL-2 secretion by frozen MNCs is independent of IL-1, and (3) inactivation of PGE-2-secreting cells during the freezing procedure is responsible for increased IL-2 secretion. Elimination of CD8+ T cells (putative suppressor cells) from MNCs has also resulted in the production of increased amounts of IL-2 by fresh cells, and again, freezing did not further enhance the IL-2-secreting activities of MNCs, that are devoid of CD8+ T cells. This confirms that the increased IL-2 production is due to the inactivation of immuno-down-regulatory cells. The results provide further evidence that the lack of active, suppressor T cells, monocytes, and increased IL-1 and -2 production may be responsible for the previously reported enhanced immunoglobulin-producing abilities of cryopreserved cells from healthy subjects and from patients with lung cancer.     

Reduction of Adipose Tissue Mass by the Angiogenesis Inhibitor ALS-L1023 from Melissa officinalis

It has been suggested that angiogenesis modulates adipogenesis and obesity. This study was undertaken to determine whether ALS-L1023 (ALS) prepared by a two-step organic solvent fractionation from Melissa leaves, which exhibits antiangiogenic activity, can regulate adipose tissue growth. The effects of ALS on angiogenesis and extracellular matrix remodeling were measured using in vitro assays. The effects of ALS on adipose tissue growth were investigated in high fat diet-induced obese mice. ALS inhibited VEGF- and bFGF-induced endothelial cell proliferation and suppressed matrix metalloproteinase (MMP) activity in vitro. Compared to obese control mice, administration of ALS to obese mice reduced body weight gain, adipose tissue mass and adipocyte size without affecting appetite. ALS treatment decreased blood vessel density and MMP activity in adipose tissues. ALS reduced the mRNA levels of angiogenic factors (VEGF-A and FGF-2) and MMPs (MMP-2 and MMP-9), whereas ALS increased the mRNA levels of angiogenic inhibitors (TSP-1, TIMP-1, and TIMP-2) in adipose tissues. The protein levels of VEGF, MMP-2 and MMP-9 were also decreased by ALS in adipose tissue. Metabolic changes in plasma lipids, liver triglycerides, and hepatic expression of fatty acid oxidation genes occurred during ALS-induced weight loss. These results suggest that ALS, which has antiangiogenic and MMP inhibitory activities, reduces adipose tissue mass in nutritionally obese mice, demonstrating that adipose tissue growth can be regulated by angiogenesis inhibitors.     

Cryopreservation of ovarian tissues temporarily suppresses the proliferation of granulosa cells in mouse preantral follicles

Cryopreservation of ovarian tissue has been reported to delay the development of preantral follicles during in vitro culture, but the mechanism causing this impairment has not been brought to light. In order to elucidate the underlying mechanism of delayed follicular development, we evaluated the effects of cryopreservation on the proliferation of granulosa cells during culture of mouse preantral follicles, as a sufficient population of granulosa cells is critical for normal follicular development. Additionally the initial cell death of granulosa cells was estimated immediately after cryopreservation. The ovarian tissues obtained from 12-day-old female mice were cryopreservation by vitrification. The granulosa cell proliferation was evaluated by measuring the PCNA expression and the expression of cell cycle regulators such as cyclin D2, CDK4, cyclin E and CDK2 in preantral follicles isolated from fresh and cryopreserved ovarian tissues that were cultured for 48 h. The viability of granulosa cells was evaluated by measuring the proportion of necrotic areas. The granulosa cell proliferation of the cryopreserved preantral follicles was decreased significantly compared to that of the fresh controls at 0 and 24 h after culture (P < 0.05), and this was increased to the control levels after 48 h of culture. The expressions of cyclin D2, Cdk 4, cyclin E and Cdk2 were also decreased in the cryopreserved ovarian tissues at 0 and 24 h after culture (P < 0.05), but they were increased to the control levels after 48 h of culture. The proportion of the necrotic area was significantly higher in cryopreserved preantral follicles compared to that of the fresh preantral follicles (P < 0.05). This suggests that cryopreservation of ovarian tissues may delay the preantral follicle development by temporary suppressing the granulosa cell proliferation through the cell cycle regulators (cyclin D2, Cdk4, cyclin E and Cdk2) and by granulosa cell death immediately after warming.     

The cryopreservation of high concentrated PBMC for dendritic cell (DC)-based cancer immunotherapy

Peripheral blood mononuclear cells (PBMC) have been accepted as a unique material for cancer immunotherapy using dendritic cells (DC) or activated lymphocytes that are being developed as an alternative or adjuvant to conventional therapies such as surgery, chemotherapy and radiation treatment. Although successful cryopreservation of large numbers of PBMC is critical for the immunotherapy, subsequent functional study of the effects of PBMC cryopreservation on differentiation into immune cells has not been well defined. In this study, over 1.0 × 10⁸ cells/ml PBMC were cryopreserved as long as 52 weeks using a controlled-rate freezer (CRF) and stored in a vapor phase of liquid nitrogen tank. The effect of PBMC cryopreservation on differentiation into DC was studied by comparing the phenotypic and functional properties of immature DC (iDC) and mature DC (mDC) derived from cryopreserved PBMC to those from fresh PBMC. The results show that cryopreservation of PBMC at a fairly high cell concentration does not significantly affect cell recovery, viability, or phenotypes of PBMC. After differentiation into DC, iDC and mDC derived from cryopreserved PBMC had their typical phenotypes and function equivalent to those derived from fresh PBMC. Therefore, the improved cryopreservation process of PBMC described in this study is available for DC-based cancer immunotherapy.