Immobilization of Cryopreserved Primary Rat Hepatocytes for the Development of a Bioartificial Liver System

Primary rat hepatocytes were cryopreserved in hormonally-defined medium (HDM) containing 40% (v/v) fetal bovine serum (FBS) and 10% (v/v) dimethyl sulfoxide (DMSO) in liquid N₂ for 6 months. After thawing, the cells were immobilized using 2% (w/v) alginate and 0.5% (w/v) chitosan solutions. The capacities of ammonia removal and urea synthesis of the immobilized-thawed hepatocytes were similar to those of immobilized hepatocytes without cryopreservation. This result shows that immobilized hepatocytes after cryopreservation are useful for the development of a bioartificial liver system.     

Neuropeptides to replace serum in cryopreservation of mesenchymal stromal cells?

Background aimsThe therapeutic potential of human mesenchymal stromal cells (MSCs) has generated considerable interest in a wide variety of areas. MSC banking is feasible, but the optimal technique of cryopreservation remains to be determined.MethodsTo reduce dimethyl sulfoxide (DMSO) concentration in cryopreservation medium, DMSO was replaced with sucrose or trehalose. To increase cell survival and proliferation rates after thawing and to eliminate the need for fetal bovine serum (FBS), neuropeptides of the vasoactive intestinal peptide/glucose-dependent insulinotropic peptide/pituitary adenylate cyclase activating polypeptide family were added to the cryopreservation medium. Cell survival was analyzed by a trypan blue dye exclusion assay. Cell proliferation of cryopreserved MSCs was determined after 7 days of culture.ResultsNo significant differences in cell survival rates were detected between cryopreservation solutions with 5% and 10% DMSO, independently of the addition of trehalose or sucrose. Cell proliferation rates tended to be highest when MSCs were frozen in 5% DMSO + trehalose. FBS could be replaced by human albumin (HA) without loss in cell survival and proliferation potential. With FBS, the addition of neuropeptides could increase cell survival and proliferation rates. Without FBS or HA, cell survival and proliferation rates in the presence of neuropeptides were comparable to rates achieved with FBS or HA.ConclusionsClassic cryopreservation with 10% DMSO could be replaced by 5% DMSO + 30 mmol/L trehalose. FBS could be replaced by HA or neuropeptides without loss in cell survival and proliferation potential. The addition of neuropeptides in the cryopreservation medium containing FBS could increase the cell proliferation rate and consequently cellular output.     

Evaluation of cell viability and apoptosis in human amniotic fluid-derived stem cells with natural cryoprotectants

A previous study demonstrated that disaccharides, antioxidants, and caspase inhibitors can be used in freezing solutions to reduce the concentration of Me₂SO from the current standard of 10% (v/v) to 5% (v/v) or 2.5% and to eliminate fetal bovine serum (FBS) for the cryopreservation of human amniotic fluid-derived stem cells (AFSCs). Hence, this study investigated whether an irreversible inhibitor of caspase enzymes, benzyloxycarbonyl-Val-Ala-dl-Asp-fluoromethylketone (zVAD-fmk), could be used in post-thaw culture media to increase the survival rate of AFSCs. Our results showed that AFSCs cryopreserved in freezing solution containing trehalose, catalase, and 5% (v/v) Me₂SO and then supplemented with zVAD-fmk in the post-thaw culture media showed similar post-thawing viability, proliferation, and apoptosis than cells cryopreserved in the control solution (10% (v/v) Me₂SO and 20% FBS). The caspase-3 activity in all the cryopreservation solutions tested was similar to that of the control. Caspase-3, caspase-8, caspase-9, and PARP expression was not found in the cryopreserved cells. In addition, no difference was found in the survival rate and apoptosis between short-term (3 weeks) and long-term (1 year) storage of AFSCs cryopreserved in the solutions used in this study. The results of the present study demonstrate that recovery of cryopreserved cells was enhanced by using a caspase inhibitor in the post-thaw culture media.     

Cryopreservation of adult cervid testes

Several species of cervids are currently classified as threatened or endangered due to a rapid decline in their populations. Sperm cryopreservation, in association with assisted reproductive technologies, can find application for the conservation of endangered cervids. In cases of unsuccessful sperm retrieval through other means prior to the death of the animal, adult testis is the only source of sperm. Recovery of viable sperm from adult testes depends on the effective preservation of testicular tissues through optimization of cryopreservation protocols. The present study evaluated combinations of 10% dimethyl sulfoxide (DMSO) with 0% or 80% fetal bovine serum (FBS) and 20% DMSO with 0 or 20% FBS for the cryopreservation of testicular tissues of three adult cervids using uncontrolled slow freezing protocol. The cryopreserved testis was compared to chilled tissue without cryoprotectants. Results revealed that testicular tissues of barking deer cryopreserved in 20% DMSO (D20) had all the analyzed 7 parameters (number of TNP1-, PRM2 and acrosin-expressing cells/tubule and, the number of viable, morphologically normal, acrosome intact, Annexin V-negative sperm) comparable to the chilled testis. However, testicular tissues of sambhar and hog deer cryopreserved only in D20S20 had 5 of 7 parameters comparable to the chilled testis. In conclusion, D20 is acceptable for cryopreservation of barking deer and D20S20 for sambar and hog deer testes.     

Cryopreservation of viable hepatocyte monolayers in cryoprotectant media with high serum content: metabolism of testosterone and kaempherol post-cryopreservation

Little work in the literature focuses on the cryopreservation of primary hepatocytes as monolayer cultures, yet this technique offers many distinct advantages over other cryopreservation systems, including high recovery, high post-thaw nutrient penetration, and low numbers of trapped dead cells. This article investigates the cryopreservation of primary rat hepatocytes at -78 degrees C attached as monolayers to collagen coated culture dishes, and describes efforts to increase post-thaw viability and function through manipulation of the freeze/thaw protocol. Different concentrations of foetal calf serum (FCS) with 10% (v/v) dimethyl sulphoxide (ME2SO) were tested as cryopreservation media, and high cryoprotectant serum levels were found to be important in maintaining membrane integrity and function in the cryopreserved rat hepatocyte monolayer cultures. Cultures cryopreserved with 90% (v/v) FCS plus 10% (v/v) ME2SO maintain 79.7+/-6.5% of the monolayer area as viable cells with normal morphology (by image analysis), 112.7+/-14.2% protein concentration, 55.4+/-4.2% carboxyfluorescein diacetate de-acetylation, 27.2+/-7.5% kaempherol glucuronidation (a measure of UDP-glucuronosyl transferase activity), and 39.3+/-7.3% testosterone hydroxylation (a measure of cytochrome P-450 activity) compared with non-cryopreserved controls. This method of cryopreservation may provide a simple, convenient means of long-term storage of hepatocytes for in vitro metabolism studies.     

Effects of cryopreservation methods on post-thaw motility of spermatozoa from the Japanese pearl oyster, Pinctada fucata martensii

In order to develop cryopreservation techniques for Japanese pearl oyster spermatozoa, the effects of various cryopreservation conditions on post-thaw motility were examined. Spermatozoa cryopreserved with 10% methanol (MET), dimethylformamide or dimethylacetamide plus 90% diluent comprising 80% seawater and 20% fetal bovine serum (FBS) showed higher percentages of post-thaw motility than those cryopreserved with 10% dimethylsulfoxide or glycerol. When spermatozoa were cryopreserved with various concentrations (0-20%) of MET and 100-80% diluent, 10% MET showed the highest percentages of post-thaw motility. When spermatozoa were cryopreserved with 10% MET and 90% diluent comprising various concentrations (0-100%) of FBS or Ringer solution mixed with seawater, the percentages of post-thaw motility peaked at 20% FBS or Ringer solution, and were significantly higher for 20% FBS than for 20% Ringer solution. The percentages of post-thaw motility increased with increasing dilution ratios from 2.5- to 50-fold. Spermatozoa cooled to -50 degrees C and then immersed in liquid nitrogen (LN) showed higher post-thaw motility than those cooled to -30 degrees C or -40 degrees C. When spermatozoa were cryopreserved to -50 degrees C at various cooling rates by changing the sample height above the LN surface, the post-thaw motilities of spermatozoa cooled at 10 cm (cooling rate: -21.3 degrees C/min) and 12.5 cm (-15.6 degrees C/min) from the LN surface were higher than those at 5, 7.5 or 15 cm. These results indicate that 10% MET plus 90% diluent comprising 80% seawater and 20% FBS is a suitable extender for cryopreservation of Japanese pearl oyster spermatozoa and that samples should be cooled to -50 degrees C at a cooling rate between -15 and -20 degrees C/min for efficient storage.     

Proliferation and differentiation potential of cryopreserved human skin-derived precursors

Objectives: Skin‐derived precursors are recognized to be a potentially autologous and accessible source of neural precursor cells for drug screening or cell‐based treatments, in many neurological disorders. Thus, it is necessary to investigate appropriate methods for cryopreservation of such human skin‐derived precursors (hSKPs). The aim of this study was to evaluate different cryopreservation techniques for retention of hSKPs to discover an optimized protocol. Materials and methods: We cryopreserved hSKPs treated with 0%, 10%, 20%, 30% and 40% foetal bovine serum (FBS) and three concentrations of dimethylsulphoxide (DMSO) 5%, 10% and 15%, with two different storage periods in liquid nitrogen (2 days: short‐term storage; and 2 months: long‐term storage). Then, we assessed survival and proliferation levels of the cells after freeze–thaw processes, by viability measurement and colony‐forming assay. For detecting hSKPs, we used immunocytochemistry and RT‐PCR assessments. Results: Our findings indicated that hSKPs cryopreserved in 5% DMSO without FBS, had better survival and proliferation potentials compared to other working formulations. With various concentrations of cryoprotectants over different time periods, hSKPs retained their differentiation potentiality and were able to differentiate into neurons (NFM and βΙΙΙ tubulin‐positive), glial cells (GFAP‐positive) and smooth muscle cells (SMA‐positive). Conclusions: Results revealed that in only 5% DMSO, hSKPs could be cryopreserved for long‐term storage with considerable survival and proliferation levels, without losing multipotency.     

海藻酸微囊替代DMSO和FBS的STO细胞冷冻保存研穷

目的：改进现有的细胞冷冻保存方法,建立一个不舍二甲基亚砜（DMSO）和血清（FBS）的高效冷冻保存方法,为细胞治疗等临床实践提供优质细胞。方法：海藻酸微囊包埋鼠胚成纤维细胞（STO细胞）后用不含DMSO和FBS的冷冻保存液进行冷冻保存。,设四个对照组：添加10％DMSO和20％FBS的组、仅添加10％DMSO的组、仅添加20％FBS、DMSO和FBS均不添加组。在冷冻前后对各实验组细胞用台盼兰染色,进行细胞计数,计算细胞存活率,同时利用溴乙锭的二聚物（EthD）、钙黄绿素-AM（Calcein—AM）进行染色观察细胞的形态,且进一步验证细胞存活率;解冻复苏后用MTT法评估细胞的增殖速度和生长活力。结果：冷冻保存30天后对各组的细胞数量、细胞存活率、细胞形态和解冻复苏后细胞的生长活力进行比较发现,海藻酸微囊包埋冷冻组的细胞数、细胞存活率、细胞形态和生长活力均与添加DMSO和FBS的组之间无显著性差异,而与其它三个对照组呈显著性差异。结论：使用海藻酸微囊替代DMSO和FBS保存STO细胞,能有效的维持细胞形态、数量、存活率,同时不影响细胞的生长活力,从而建立了一个不含DMS0和FBS的高效冷冻保存方法。     

Cryopreservation of the sperm of the Japanese bitterling

Sperm of the Japanese bitterling Tanakia limbata that had been cryopreserved with 5 or 10% methanol plus 95 or 90% foetal bovine serum (FBS) showed higher percentage and longer duration of motility than those that had been cryopreserved with 90% FBS and 10% DMSO, glycerol, N,N-dimethylacetamide or N, N-dimethylformamide. Foetal bovine serum, used as extender, had some cryoprotective effects when spermatozoa were cooled either with 10% methanol or without methanol. Spermatozoa, cooled to −40° C and then immersed in liquid nitrogen, had greater post-thaw motility than those cooled to −20, −60, or −80° C. The post-thaw percentage of motile spermatozoa increased significantly ( P < 0·001) with decreases in the freezing rate from 60 to 5°C min⁻¹. These results indicate that 10% methanol plus 90% foetal bovine serum is a suitable diluent for cryopreservation of bitterling spermatozoa and that samples should be cooled to -40°C at a low freezing rate for effective storage.     

Cryopreservation of chum salmon blastomeres by the straw method

In order to preserve genetic resources of chum salmon, Oncorhynchus keta, optimum conditions for cryopreservation of isolated blastomeres were investigated. Survival rates under various conditions were compared: the nature and the concentration of cryoprotectants before and after freezing, the seeding temperature, and the developmental stages of donor embryos. Isolated blastomeres immersed for 30 min in Eagle's MEM containing both a cryoprotectant and 10% fetal bovine serum (FBS) at 10 degrees C were transferred into a straw and frozen at 1 degrees C/min to -30 degrees C by a programmable freezer before being plunged into liquid nitrogen. Ice seeding was carried out at -5 to -15 degrees C. Frozen blastomeres were thawed in water at 15 degrees C. Blastomeres cryopreserved with MEM containing 10% dimethyl sulfoxide (Me(2)SO) and 10% FBS (10% Me(2)SO/MEM10) showed higher survival rates than those cryopreserved with MEM containing 10% FBS and 10% glycerol, ethyleneglycol, 1, 2-propanediol, or sucrose. Blastomeres treated with 10% Me(2)SO/MEM10 showed higher survival rates than those treated with MEM containing only 10% Me(2)SO. Blastomeres seeded above -10 degrees C showed higher survival rates than non-seeded ones. Frozen blastomeres at advanced stages demonstrated high survival rates. Blastomeres cryopreserved under optimum conditions showed survival rates of 59.3+/-2.8%. These results indicate that 10% Me(2)SO/MEM10 is a suitable cryoprotectant medium to cryopreserve chum salmon blastomeres, that seeding should be carried out above -10 degrees C on pre-freezing, and that blastomeres at the blastula stage should be used as material.     

Incorporation of DMSO and dextran-40 into a gelatin/alginate hydrogel for controlled assembled cell cryopreservation

A new cell cryopreservation strategy for cell-assembling constructs was proposed. With this strategy, different concentrations of dimethysulfoxide (DMSO) and dextran-40 were directly incorporated into the cell/gelatin/alginate systems, prototyped according to a predesigned structure, cryopreserved at −80 °C for 10 days and followed a thawing process at 17 °C. The rheological properties, bonding water contents and melting points of the gelatin/alginate hydrogel systems were changed with the addition of different amounts of DMSO. The microscopy analysis, (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrasodium bromide (MTT) and hematoxylin and eosin (HE) staining indicated that the cell numbers were progressively in a selected DMSO concentration range. With DMSO 5% (v/v) alone, the metabolic rate in the construct attained (81.3 ± 5.7)%. A synergistic effect was achieved with the combination of the DMSO/gelatin/alginate and dextran-40/gelatin/alginate hydrogel systems. These results indicated that the inclusion of DMSO and dextran-40 in the hydrogel could effectively enhance the cell preservation effects. This cryopreservation strategy holds the ability to be widely used in organ manufacturing techniques.     

Comparison of stem cell viability of bone marrow cryopreserved by two different methods

Two different cryogenic methods were used to study the preservation of murine bone marrow cells. Compared to the classical methods, in which separated mononuclear marrow cells in 10% dimethyl sulfoxide (DMSO) were cryopreserved in liquid nitrogen (-196 degrees C), a modified technique was carried out by cryopreservation of unfractionated marrow cells in a mixed protectant of 5% DMSO and 6% hydroxyethyl starch (HES) at -80 degrees C. Samples that were separately thawed after storage for 1, 4, 8, and 12 weeks were assayed for cell viability and recovery of CFU-GM and CFU-S. No macroscopic clumping of cells was noted either in fractionated or in unfractionated marrow cell cryopreservations. A mild damage, about 25% reduction of stem cells, was found at 1 week and did not deepen further. It seems that the greatest loss of stem cells occurred in the process of cryopreservation itself. Compared to prefreeze values, both a high number of cells that excluded trypan blue (87 +/- 3.4%) and a high recovery of CFU-GM (75 +/- 9.8%) and CFU-S (74 +/- 11.2) were observed in unfractionated marrow samples cryopreserved with the DMSO/HES mixture at -80 degrees C for 3 months and these results were very similar to those obtained from fractionated mononuclear marrow cells cryopreserved at -196 degrees C. The DMSO/HES protectant provides a simplified bone marrow cryopreservation technique that should be favorable to clinical application because of its high stem cell recovery and avoidance of cell-separation manipulation.     

The viability of cryopreserved PBPC depends on the DMSO concentration and the concentration of nucleated cells in the graft

BACKGROUND: DMSO is widely used as a cryoprotectant for PBPC. It is desirable to reduce the amount of DMSO without jeopardizing the quality of the stem cell product. The present study was undertaken to investigate whether recovery and survival of CD34+ cells would be significantly altered when PBPC used for autologous transplantations were cryopreserved with four different DMSO concentrations. METHODS: Apheresis samples of PBPC from 20 consecutive patients were mixed in parallel with 2%, 4%, 5% and 10% DMSO, frozen with identical cell concentrations at a controlled rate, and stored in liquid nitrogen for 6-8 weeks. PBPC samples from 11 consecutive patients were also cryopreserved with two different cell concentrations (150 and 300 x 10(6) nucleated cells/mL) to investigate the effect of increasing the cell concentrations while decreasing the DMSO concentration. The flow cytometric absolute count method, based on ISHAGE guidelines, was used to measure the absolute count of total and viable CD34+ cells in the post-thaw samples. RESULTS: PBPC cryopreserved at 150 x 10(6) cells/mL with 2% DMSO yielded significantly inferior CD34+ cell recovery (P < 0.001) and survival (P < 0.001) compared with cryopreservation with 4% and 5% DMSO. This was also observed when comparing higher cell concentrations. However, a reduced cell survival (P = 0.02) was observed when the nucleated cell concentration was increased from 150 to 300 x 10(6) cells/mL in samples cryopreserved with 5% DMSO. DISCUSSION: We conclude that 5% DMSO may be the optimal dose for cryopreserving PBPC as long as the cells have not been concentrated at much more than 200 x 10(6) nucleated cells/mL.     

Manual cryopreservation of human alveolar periosteal tissue segments: Effects of pre-culture on recovery rate

Cultured human periosteal sheets constitute a promising grafting material for periodontal tissue regenerative therapy. However, preparation of these sheets usually requires six weeks or longer, and this lengthy commitment and delay limits both clinical applicability and availability. The aim of this study is to develop an efficient, practical, cost-effective cryopreservation method for periosteal tissue segments (PTSs). Human PTSs were aseptically excised from alveolar bone and pre-cultured in Medium 199 + 10% fetal bovine serum (FBS) for the indicated number of days before they were slowly frozen down to −75 °C in a commercial freezing vessel using medium containing 10% dimethyl sulfoxide (Me₂SO) and various concentrations of FBS. After fast-thawing at 37 °C, PTSs were again cultured, and their growth and responses to standard osteogenic induction were evaluated (vs. freshly excised PTSs). Proliferating cells were obtained at the highest levels from cryopreserved PTSs that were pre-cultured for 14 days before freezing. When a concentration of 50% or more FBS was included in the cryopreservation solution, cells migrated out more actively and grew faster. Importantly, osteoinduction up-regulated alkaline phosphatase (ALP) activity and osteoblastic marker mRNAs in cryopreserved PTS-derived sheets just as effectively as it did in native PTS-derived ones. These data suggest that pre-conditioned PTSs can be efficiently cryopreserved in a freezing solution containing high FBS by traditional manual cryopreservation methods without aid of a program freezer or more elaborate equipment.     

Optimal conditions for heart cell cryopreservation for transplantation

Cultured myocyte transplantation into an infarcted myocardium has been shown to improve contractile function. Cryopreservation of cultured muscle cells or heart tissue will be important for the technology to be practical. This study, using fetal cardiomyocytes, evaluated the optimal conditions for muscle cell cryopreservation. Study 1: Fetal rat cardiomyocytes were isolated and cultured. The freshly isolated and passage 1, 2, 3 and 4 cells were cryopreserved in a solution containing 70% IMDM, 20% FBS and 10% DMSO and stored in –196°C for 1, 2, 4, 8, 12 and 24 weeks. The cells were thawed and cultured. Cell number and contractility were evaluated at 0, 2, 4, 6, 8 and 10 days of culture. Study 2: Rat myocardium was cryopreserved in sizes of 0.2, 2 and 6 mm³ for 1 week. The tissue was thawed and cells were isolated. Cell growth and contractility were evaluated. (1) Cardiomyocytes grew and contracted after cryopreservation. Storage time did not affect cell survival rate, beating cell numbers and beating rates. Increasing cell passage prior to cryopreservation decreased the percentage of beating cells. (2) Cells isolated from cryopreserved tissue grew in vitro and contracted normally. Cell yield decreased with increased cryopreserved tissue size. Fetal rat cardiomyocytes survived and functioned after in vitro cryopreservation. Viable cells can be isolated from cryopreserved myocardium and cultured. Cryopreservation of small pieces of myocardium is preferred for maximal cell yields.     

Development of a cryopreservation protocol for long-term storage of black tiger shrimp (Penaeus monodon) spermatophores

The objectives of this study were to determine the effect of cryoprotectants on sperm viability and develop a freezing protocol for long-term storage of P. monodon spermatophores. Spermatophores suspended for 30 min in calcium-free saline (Ca-F saline) containing the cryoprotectants dimethyl sulfoxide (DMSO), ethylene glycol (EG), 1,2-propylene glycol (PG), formamide, and methanol at concentrations of 5, 10, 15, or 20% were studied using a modified eosin-nigrosin staining technique. The smallest reductions in apparent sperm viability occurred with DMSO; therefore, a freezing protocol was developed using Ca-F saline containing 5% DMSO. Spermatophores were cryopreserved using three protocols; cooling to a final temperature of -30, -80 or -80 degrees C and immediately stored in liquid nitrogen (cooling rates of -2, -4, -6, -8, -10, -12, -14 or -16 degrees C/min). Frozen spermatophores were thawed (2 min) at 30, 60, 70, or 90 degrees C. Successful cryopreservation of spermatophores in liquid nitrogen was achieved by a one-step cooling rate of -2 degrees C/min between 25 and -80 degrees C before storing in liquid nitrogen. Optimal thawing was in a 30 degrees C water bath for 2 min; this yielded live sperm after storage in liquid nitrogen for 210 days. Average sperm viability for fresh (97.8+/-2.9%) and cryopreserved spermatophores held for less than 60 days (87.3+/-4.1%) did not differ (P>0.05); however, that for spermatophores stored in liquid nitrogen between 90 and 210 days were lower (P<0.05) and varied from 27.3+/-3.4 to 53.3+/-4.3%. Thawed spermatophores previously held in liquid nitrogen for less than 62 days fertilized eggs (fertilization and hatching rates of 71.6-72.2% and 63.6-64.1%, respectively) at rates comparable to fresh spermatophores (70.8-78.2% and 66.3-67.8%, respectively). In conclusion, sperm within cryopreserved spermatophores stored in liquid nitrogen retained their viability for up to 210 days.     

Strategies for the cryopreservation of microencapsulated cells

The major challenge in developing cryopreservation protocols for microencapsulated cells is that the relatively large size (300-400 microm) and the fragile semipermeable membrane of microcapsules makes them particularly prone to cryodamage. Rapid-cooling cryopreservation protocols with high DMSO concentrations (3.5M, 25% v/v) resulted in low post-thaw cell viability (<10%), which did not improve with higher concentrations (4.5M, 32% v/v) and longer exposure to DMSO, even though the majority of microcapsules (60-80%) remained intact. Subsequent investigations of slow cooling with a range of DMSO and EG concentrations resulted in a much higher post-thaw cell viability (80-85%), with the majority of the microcapsules remaining intact ( approximately 60%) when DMSO was used at a concentration of 2.8M (20% v/v) and EG at a concentration of 2.7M (15% v/v). The presence of 0.25M sucrose significantly improved post-thaw cell viability upon slow cooling with 2.8M (20% v/v) DMSO, although it had no effect on microcapsule integrity. Multistep exposure and removal of sucrose did not significantly improve either post-thaw cell viability or microcapsule integrity, compared to a single-step protocol. Ficoll 20% (w/v) also did not significantly improve post-thaw cell viability and microcapsule integrity. Hence, the optimal condition for microcapsule cryopreservation developed in this study is slow cooling with 2.8M (20% v/v) DMSO and 0.25M sucrose.     

Cryopreservation of nucellar cells of navel orange (Citrus sinensis Osb. var. brasiliensis Tanaka) by vitrification

The nucellar cells of navel orange(Citrus sinensis Osb. var. brasiliensis Tanaka) were successfully cryopreserved by vitrification. In this method, cells were sufficiently dehydrated with highly concentrated cryoprotective solution(PVS2) prior to direct plunge in liquid nitrogen. The PVS2 contains(w/v) 30% glycerol, 15% ethylene glycol and 15% DMSO in Murashige-Tucker medium(MT) containing 0.15 M sucrose. Cells were treated with 60% PVS2 at 25°C for 5 min and then chilled PVS2 at 0°C for 3 min. The cell suspension of about 0.1 ml was loaded in a 0.5 ml transparent plastic straw and directly plunged in liquid nitrogen for 30 min. After rapid warming, the cell suspension was expelled in 2 ml of MT medium containing 1.2 M sucrose. The average rate of survival was about 80%. The vitrified cells regenerated plantlets. This method is very simple and the time required for cryopreservation is only about 10 min.Abbreviations DMSO dimethyl sulfoxide - PVS2 vitrification solution - LN liquid nitrogen - DSC differential scanning calorimeter - BA 6-benzylaminopurine - MT Murashige-Tucker basal medium - INAA naphthaleneacetic acid     

Cryopreservation of amniotic fluid-derived stem cells using natural cryoprotectants and low concentrations of dimethylsulfoxide

Amniotic fluid-derived stem cells (AFSCs) are a potential cell source for therapeutic applications. They can be easily mass produced, cryopreserved and shipped to clinics for immediate use. However, one major obstacle to the manufacturing of clinical grade stem cells is the need for current good manufacturing practices for cryopreservation, storage, and distribution of these cells. Most current cryopreservation methods used for stem cells include the potentially toxic cryoprotectant (CPA) dimethylsulfoxide (Me₂SO) in the presence of animal serum proteins that prevent direct use of these cells in human therapeutic applications. To avoid any potential cryoprotectant related complications, it will be essential to develop non-toxic CPAs or reduce CPA concentration in the freezing media used. In this study, we assessed the use of disaccharides, antioxidants and caspase inhibitors for cryopreservation of AFSCs in combination with a reduced concentration of Me₂SO. The thawed cells were tested for viability with MTT assays and a growth curve was created to measure population doubling time. In addition, we performed flow cytometry analysis for cell surface antigens, RT-PCR for mRNA expression of stem cell markers, and assays to determine the myogenic differentiation potential of the cells. A statistically significant (p < 0.05) increase in post-thawed cell viability in solutions containing trehalose, catalase and _ZVAD-fmk with 5% Me₂SO was observed. The solutions containing trehalose and catalase with 5% or 2.5% (v/v) Me₂SO produced results similar to those for the control (10% (v/v) Me₂SO and 30% FBS) in terms of culture growth, expression of cell surface antigens and mRNA expression of stem cell markers in AFSCs cryopreserved for a minimum of 3 weeks. Thus, AFSCs can be cryopreserved with 1/4 the standard Me₂SO concentration with the addition of disaccharides, antioxidants and caspase inhibitors. The use of Me₂SO at low concentrations in cell freezing solutions may support the development of clinical trials of AFSCs.     

Unfractionated human marrow cell cryopreservation using dimethylsulfoxide and hydroxyethyl starch

Unfractionated bone marrow (BM) cells were cryopreserved in 1- to 2-ml aliquots using a mixture containing both 5% dimethylsulfoxide (DMSO) and 6% hydroxyethyl starch (HES) in an attempt to increase the viable cell yield and reduce the clumping after thawing, observed when 10% DMSO is used alone. Samples thawed after storage for 6 months in the vapor phase of liquid nitrogen, were assayed. Compared to prefreeze values, there was both a greater number of cells that excluded Trypan Blue (50 +/- 12 vs 28 +/- 12%, P less than .01) and a greater CFU-C Recovery (110 +/- 20 vs 89 +/- 35%, P less than .02) for cells in the DMSO/HES mixture, compared to those in 10% DMSO alone. No macroscopic clumping of the thawed cells was observed for those cryopreserved in the mixture in contrast to those in DMSO alone. Freezing was done without a rate-controlled freezing apparatus by simply placing the samples initially into a -80 degrees C freezer, and then later into a liquid nitrogen freezer. Additional samples stored in the DMSO/HES mixture were kept at only -80 degrees C, and when thawed 12 to 16 months later also gave an excellent CFU-C recovery (105 +/- 39% of prefreeze). The DMSO/HES mixture allows for a simplified BM cryopreservation technique that not only assures excellent recovery of CFU-Cs and eliminates clumping upon thawing, but also does not require either the use of a rate-controlled freezer or liquid nitrogen temperatures for storage up to a year.