In-vitro growth of murine pre-antral follicles after isolation from cryopreserved ovarian tissue

The low temperature storage of ovarian tissue allows patients at risk of premature menopause to preserve their fecundity. One strategy for harvesting mature oocytes may be to isolate small follicles from the stroma for in-vitro culture. The aim of this study was to assess the survival and growth of murine follicles during in-vitro culture after isolation from tissue frozen-thawed in various cryoprotective agents. The effect of different seeding and thawing temperatures was also investigated. Pre-antral follicles 100--135 microm in diameter were isolated from fresh and frozen-thawed tissue and cultured in vitro for 8 days. In the fresh control group 79 +/- 3% of follicles survived in-vitro culture and grew to antral stages. Fewer follicles survived after isolation from tissue cryopreserved in dimethyl sulphoxide (43 +/- 5%) or propylene glycol (24 +/- 2%) and none survived freeze-thawing in glycerol. Lowering the seeding temperature from -5 degrees to -7 degrees or -9 degrees C reduced follicle survival rates from 49 +/- 4% to 26 +/- 1% and 28 +/- 3% respectively. If thawing was carried out at 27 degrees C follicle survival rate was higher (38 +/- 7%) than at 37 degrees C (26 +/- 2%) or 47 degrees C (20 +/- 6%). Follicles surviving 8 days of in-vitro culture were stimulated with human chorionic gonadotrophin. The number of mature oocytes released did not differ between any experimental group. The results indicate that follicles isolated from frozen-thawed tissue can be grown to antral sizes and produce mature oocytes. The in-vitro culture system also proved a sensitive method for testing variations in the freeze-thaw protocol.     

Survival of chlamydiae after cooling to -196 degrees C.

Factors influencing the survival of chlamydiae after freezing were reexamined. From the data presented, it is suggested that preservation of laboratory-grown chlamydiae is best achieved through the use of sucrose as the cryoprotective agent, in the presence of 10% serum. Dimethyl sulfoxide and glycerol are more toxic. The period of exposure to sucrose before freezing must be kept as short as possible and be at 4 degrees C rather than at room temperature. The rate of cooling during freezing in sucrose is not important; however, cooling at a rate slower than 1 degrees C/min should be avoided. Since chlamydial survival is increased by rapid thawing, the volume of the sample should be kept to a minimum. Thawed suspensions should be inoculated onto cell monolayers without delay. The application of these methods may increase the proportion of stored clinical specimens in which chlamydiae can be found.     

Cryoprotective agent and temperature effects on human sperm membrane permeabilities: convergence of theoretical and empirical approaches for optimal cryopreservation methods

Previous reports have left unresolved discrepancies between human sperm cryopreservation methods developed using theoretical optimization approaches and those developed empirically. This study was designed to investigate possible reasons for the discrepancies. Human spermatozoa were exposed to 1 mol/l glycerol, 1 mol/l dimethyl sulphoxide (DMSO), 1 mol/l propylene glycol (PG) or 2 mol/l ethylene glycol (EG) at 22, 11 and 0 degrees C, then returned to isosmotic media while changes in cell volume were monitored. Activation energies (E(a)) of the hydraulic conductivity (L(p)) in the presence of cryoprotective agents (CPA) (L(p)(CPA)) were 22.2 (DMSO), 11.9 (glycerol), 15.8 (PG), and 7.8 (EG) kcal/mol. The E(a) values of the membrane permeability to CPA (P(CPA)) were 12.1 (DMSO), 10.4 (glycerol), 8.6 (PG) and 8.0 (EG) kcal/mol. These data indicated that even at low temperatures, EG permeates fastest. The high L(p)(CPA) in the presence of EG and low associated E(a) would allow spermatozoa to remain closer to equilibrium with the extracellular solution during slow cooling in the presence of ice. Collectively, these data suggest that the increase of the E(a) of L(p) in the presence of CPA at low temperature is the likely reason for the observed discrepancy between theoretical predictions of spermatozoa freezing response and empirical data.     

Determination of optimal cryoprotectants and procedures for their addition and removal from human spermatozoa

The objective was to test the hypothesis that the optimal cryoprotective agent for cryopreservation of human spermatozoa would be a solute for which cells have the highest plasma membrane permeability, resulting in the least amount of volume excursion during its addition and removal. To test this hypothesis, theoretical simulations were performed using membrane permeability coefficients to predict optimal procedures for the addition and removal of a cryoprotectant. Simulations were performed using data from four different cryoprotectants: (i) glycerol, (ii) dimethyl sulphoxide, (iii) propylene glycol and (iv) ethylene glycol. Thermodynamic formulations were applied to determine approaches for the addition and removal of 1 M and 2 M final concentrations of cryoprotectant, allowing the spermatozoa to maintain a cell volume within their osmotic tolerance limits. Based on these data, ethylene glycol was predicted to be optimal for minimizing volume excursions among the solutes evaluated. These predictions were then experimentally tested using glycerol as the control cryoprotectant and ethylene glycol as the experimental cryoprotectant. The results indicate that there was a higher (P < 0.05) recovery of motile spermatozoa after cryopreservation when using 1 M ethylene glycol than with 1 M glycerol, supporting the hypothesis that use of the cryoprotectant for which the cell has the highest permeability will result in higher cell survival.      

Effect of freezing rate and exposure time to cryoprotectant on the development of mouse pronuclear stage embryos.

BACKGROUND: The effects of exposure time (20 versus 45 s) to a high concentration of cryoprotectant (7.0 mol/l ethylene glycol with 0.5 mol/l sucrose) and freezing rates (1200-10 300 degrees C/min) during rapid freezing of mouse pronuclear stage embryos on survival and development to blastocysts were investigated. Different freezing rates were achieved by directly plunging the straws (rapid freezing) and open pulled straws (OPS) in liquid nitrogen (OPS freezing) and by plunging the straws (super rapid) and OPS (super OPS) in a super cooled liquid nitrogen chamber (at -212 degrees C) before storage in liquid nitrogen. METHODS: Morphologically intact mouse zygotes (n = 891) pre-equilibrated in 1.5 mol/l ethylene glycol for 5 min were either loaded in 0.25 ml straws containing cryoprotectant or loaded in OPS with 2 microl cryoprotectant. After 20 or 45 s of loading the straws or mixing in cryoprotectant and loading in OPS, they were plunged either directly in to liquid nitrogen or were plunged first in to liquid nitrogen in a super cooled chamber and then stored in liquid nitrogen. Zygotes were thawed and intact embryos cultured in vitro. RESULTS: The rate of survival was higher (91%, P < 0.01) when zygotes were frozen with rapid freezing compared with super rapid, OPS and super OPS freezing rates with an exposure time of 20 s (70, 65, and 76% respectively). When zygotes were exposed to cryoprotectant for 45 s and frozen with rapid freezing rates, the survival was higher (86%, P < 0.01) compared with those frozen with OPS (62%) but was not different from those frozen with super rapid and super OPS freezing rates (81 and 75%). A higher rate of survival was observed when zygotes were exposed to cryoprotectant for 45 s and frozen with super OPS than with OPS freezing (75 versus 62%; P < 0.05). The rate of cleavage and development of intact zygotes to blastocysts was not different among the different groups. CONCLUSION: Exposure of zygotes to a high concentration of cryoprotectant (7.0 mol/l ethylene glycol with 0.5 mol/l sucrose) for 20 or 45 s did not influence their survival and development and increasing the freezing rate from 1200-10 300 degrees C/min was of no advantage when using a rapid freezing procedure for freezing mouse pronuclear stage embryos.     

Vitrification of mouse germinal vesicle oocytes: effect of treatment temperature and egg yolk on chromatin and spindle normality and cumulus integrity

The success rates for cryopreservation of immature oocytes from several species including human remain low, in contrast to major improvements with mature oocytes. In this study, a new approach has been developed using a short exposure ultra-rapid freezing protocol, examining the effect of temperature and egg yolk (two factors which may be expected to influence membrane flexibility) on the cryostability of immature mouse oocytes and cumulus complexes. These two factors were tested in various patterns for their cryoprotective effect using ethylene glycol as the principal cryoprotectant. The results showed that 37 degrees C pre- and post-freeze exposure significantly improved both survival and normal spindle configuration after in-vitro maturation. Egg yolk was found to produce further beneficial effects on both the oocyte and cumulus cell integrity, with the best effects being obtained at 37 degrees C with inclusion of egg yolk both before and after the freezing. This protocol produced > 80% normal survival post-thaw with intact and attached cumulus complex, 84% maturation rate and 45% normal metaphase configuration. In summary, a unique combination of high survival and meiotic normality together with good preservation of the attached cumulus cell mass has been achieved using a simple new vitrification procedure.     

Recovery, survival and functional evaluation by transplantation of frozen-thawed mouse germ cells

BACKGROUND: Establishing a successful method for testicular stem cell transplantation of frozen-thawed testicular cells would be of immense benefit to boys with childhood cancer undergoing a sterilizing treatment. In this study, we evaluated different cryopreservation protocols in a mouse model by means of testicular germ cell transplantation (TGCT), in order to establish an optimal freezing protocol. METHODS AND RESULTS: In a first series of experiments, we compared an uncontrolled protocol with 1.5 mol/l dimethyl sulphoxide (DMSO) versus a controlled long protocol (cooling to -80 degrees C) and observed a better viability with the latter protocol (36% versus 48%, P < 0.05). We then compared survival after two thawing methods (37 degrees C water versus ice water) in either a DMSO- or an ethylene glycol (EG)-based protocol, and found no difference. In order to evaluate the functional capacity of the cryopreserved testicular suspension, TGCT was performed with both fresh and frozen-thawed suspensions. In 90% of the successfully injected testes, spermatogenesis was reinitiated using fresh suspensions. In contrast, this figure was only 12.5 and 22.7% after cryopreservation, for the short controlled EG protocol and the uncontrolled DMSO protocol, respectively. CONCLUSION: Reinitiation of spermatogenesis is possible after cryopreservation of testicular germ cell suspensions. Although cell survival was acceptable, our results after TGCT show that our protocols need further improvement.     

New method for preserving cytology specimens.

A method of freezing cytological material for long term storage, based on a modification of that used in tissue typing for the storage of lymphocytes, was developed. The method entails the centrifugation of the specimen, adding 1 ml AB or fetal calf serum and 1 ml 20% dimethyl sulphoxide aliquoted into 2 ml tubes, and storage at -70 degrees C. Cytological detail was well preserved in a variety of samples including fine needle aspirates, urine, and bronchial brushings. Three dimensional architecture was also preserved. The method is practical, easy to perform, and allows retrospective studies to be undertaken with the subsequent use of a variety of special staining techniques, including immunocytochemistry.     

Ultrarapid embryo freezing: effect of dissolved gas and pH of the freezing solutions and straw irradiation

While optimizing the ultrarapid embryo freezing procedure wenoted that embryo survival was lowest when gas bubbles formedin the straws. Here we report the influences of gassing thefreezing solutions with 5% CO₂ in air, degassing the solution,the pH of the medium and straw irradiation on the survival anddevelopment in vitro of 2-cell mouse embryos. Embryos were ultrarapidlyfrozen in medium M2 containing 3 M dimethyl sulphoxide and 0.25M sucrose. Significantly fewer embryos survived freezing andthawing in gassed solutions. The subsequent development of intactembryos to blastocysts was similar in gassed and non-gassedsolutions. Survival and development of embryos frozen in solutionsof pH 7.0, 7.6 or 8.0 was similar, but fewer embryos developedto blastocysts after freezing in media at pH 6.0. In strawssterilized by gamma irradiation before use, embryo survivalafter freezing and thawing was dramatically reduced comparedwith that seen when non-irradiated straws were used. With non-gassedsolutions significantly more gas bubbles formed in irradiatedstraws than in non-irradiated straws. Although bubble formationwas significantly reduced in all straws by using degassed freezingsolutions, this did not improve embryo survival. We concludethat under conditions normally used for ultrarapid freezing,dissolved gas and pH have less effect on embryo viability thanthe use of irradiated straws.     

五种玻璃化冻存试剂单独或组合应用对髓核细胞的毒性分析

BACKGROUND: Transplantation of allogeneic intervertebral disc can be facilitated by the cryopreservation of the intervertebral disc. But the traditional cryopreservation methods always lead to the appearing of ice crystals inside and outside the cells which can cause cellular injury. The vitrification method that can avoid the formation of ice crystals have been widely applied in the cryopreservation field. However, only a few reports have assessed the vitrified cryopreservation of the intervertebral disc, and the toxicity of cryoprotectants to the nucleus pulposus cells have not been fully explored. OBJECTIVE: To determine the order of toxicity of five commonly used cryoprotectants that are used alone or in combination to rabbit nucleus pulposus cells, and to select the optimal cryoprotectant for the vitrification of the intervertebral disc. METHODS: We chose five most commonly used cryoprotectants including dimethyl sulphoxide, formamide, ethylene glycol, propylene glycol and glycerol. Then, 5 single commonly used cryoprotectants, 10 mixed agents containing any 2 commonly used cryoprotectants, and 10 mixed agents containing any 3 commonly used cryoprotectants were formulated. Cell viability of nucleus pulposus cells was determined using cell counting kit-8 and fluorescein diacetate/propidium iodide method. All data obtained were analyzed statistically to choose the appropriate combining scheme with less toxicity. RESULTS AND CONCLUSION: The order of the toxicity of these five commonly used cryoprotectants from low to high was ethylene glycol, glycerol, formamide, dimethyl sulphoxide, and propylene glycol. The toxicity of the combined agents containing two or three commonly used cryoprotectants was lower than that of any commonly used cryoprotectants that were used to formulate them. The toxicity of the mixed agents that contained ethylene glycol or glycerol was lower than that of any other mixed agents. So we can choose the mixed cryoprotectants that contain ethylene glycol and (or) glycerol for the vitrification of the intervertebral disc.       

Cryopreservation of collagen-based tissue equivalents. II. Improved freezing in the presence of cryoprotective agents

In Part I of this study we determined an optimal cooling rate for cryopreservation of collagen-based tissue equivalents (TEs) that preserves both the postthaw cell viability and mechanical properties, but results in tissue contraction and an overall loss of opacity. The empirically determined optimal cooling rate (5 degrees C/min) was obtained in a freezing medium consisting solely of phosphate-buffered saline (PBS) at physiological concentration (1x). In the present study we report the effect of freezing on TEs in the presence of PBS and two cryoprotective agents (CPAs) (glycerol and dimethyl sulfoxide [Me(2)SO]), at two different concentrations (0.5 and 1.0 M), to two different end temperatures (-80 and -160 degrees C), at a cooling rate of 5 degrees C/min. The controlled rate freezing experiments, postthaw cell viability, and mechanical property measurements were performed as described in Part I of this study. In addition to studying the effect of CPAs on the postthaw properties of TEs, we also investigated (1). the effect of freezing TEs attached to the substrate (as opposed to detached and floating in medium) to determine differences when freezing TEs subject to static mechanical stress via a mechanical constraint to contraction; (2). the effect of freezing glutaraldehyde-fixed TEs to determine differences in freezing-mediated damage to the microstructure; and (3). the effect of freezing more mature TEs that were incubated for 4 weeks in growth factor-supplemented medium as opposed to 2 weeks in basal medium. All TEs frozen at 5 degrees C/min to -80 degrees C in the presence of 0.5 M glycerol or Me(2)SO in PBS were found to be optimally cryopreserved in terms of maintaining opacity and structure as well as cell viability and mechanical properties as compared with unfrozen TEs. The postthaw mechanical properties were adversely affected by freezing to the lower end temperature of -160 degrees C in the presence of CPAs, with the samples frozen in the 1.0 M concentration of CPAs exhibiting a total loss of structural integrity on thawing. Furthermore, TEs frozen attached to the substrate showed decreased opacity and significant contraction as compared with TEs frozen detached from the substrate, as did cross-linked samples frozen without CPA.     

Human ovarian tissue from cortex surrounding benign cysts: a model to study ovarian tissue cryopreservation

BACKGROUND: The scarcity of human ovarian tissue is a major problem in developing research on ovarian cryopreservation. We were interested in ovarian cortex surrounding benign ovarian cysts harvested during their requisite operations. METHODS: Ovarian tissue was collected from 25 women (mean age = 27.7 +/- 1.0 SEM) and frozen in serum-free cryoprotective medium. Histological and viability analysis were performed on fresh and frozen-thawed slices of tissue. RESULTS: Dermoid (n = 7), endometriosis (n = 13) and serous (n = 5) cysts were observed. Follicular densities (expressed per mm3) in ovarian cortex surrounding dermoid cysts were higher than in endometriosis and serous cysts for both histological (median of follicular densities: 13.04, 0.31 and 0.89 respectively) and viability analysis (2.93, 0.05 and 0.71 respectively). Freezing-thawing did not result in gross abnormality of follicle population either in number or morphology (80% of follicles preserved a normal pattern). However, a slight decrease of the density of living follicles (expressed per mm2) was reported. CONCLUSIONS: Ovarian cortex surrounding ovarian cysts, especially dermoid cysts, could be considered a source of ovarian tissue for future research. In our study, the cryopreservation procedure resulted in high follicular survival assessed by both histological and viability analysis. Nevertheless, further studies of in vivo and in vitro follicular maturation are needed to strengthen this model.     

卵巢组织冷冻移植在女性生育力保存中的研究进展

医疗技术的发展使得癌症患者的存活率越来越高,但放化疗治疗会导致女性卵巢功能早衰,出现过早绝经甚至丧失生育能力。卵巢组织冷冻保存是保存女性生育力的一种方式,甚至是有些患者唯一的生育力保存的选择。目前在世界范围已有17例婴儿是来自冷冻/解冻-移植后的卵巢组织,这给我们带来了希望,同时也带来了挑战,因为这项技术还存在一定的局限性。目前使用的冷冻方法可以保存卵巢内大量的始基卵泡,但是在移植的初期,因组织缺血缺氧会导致大量的卵泡丢失,影响了移植物的存活,缩减了卵巢组织在体内发挥功能的时间。卵巢组织移植存活程度的影响因素很多,包括移植前个体因素和基础疾病及治疗对卵巢的损伤,冷冻的损伤和移植组织块的大小,移植后血管再生情况等。这篇文章将对卵巢组织冷冻作为女性生育力保存方式的研究进展做一综述。     

Fertilization and development of the human oocyte following exposure to cryoprotectants, low temperatures and cryopreservation: a comparison of two techniques.

Both glycerol and dimethyl sulphoxide (DMSO) equilibration prior to cryopreservation produced adequate rates of survival and development to the hatching blastocyst stage for mouse oocytes using the two chosen cooling and warming regimes. Successful fertilization of human oocytes and further development of the embryos produced, was achieved following equilibration with DMSO at 0 degrees C. Although fertilization of fresh human ova previously exposed to glycerol was recorded, all oocytes with two pronuclei failed to continue to undergo cell division by 48 h in culture. Following cryopreservation, human oocytes were successfully inseminated after equilibration with both glycerol and DMSO. However, cell division to the 2-cell stage was recorded in only one oocyte which had undergone exposure to DMSO, freezing and thawing. No cell divisions were recorded after glycerol cryopreservation, or even after simple exposure to glycerol. Therefore it appears that DMSO and slow cooling may be the best protocol, although further evaluation will be necessary.     

Ultrastructural changes of veins in several variants of cryopreservation

The findings of ultrastructural analysis demonstrated essential changes in connective tissue cells and noncellular elements of veins after their cryopreservation at -50 degrees C and -196 degrees C. Contraction and round-up of endotheliocytes, disruption of their connections with basement membrane leading to subendothelial space widening, cytoplasm vacuolization and focal destruction of luminal plasmalemma are typical and general changes caused by cryopreservation. Dystrophy of different grades developed in connective tissues of media and adventitia. Deeper freezing negatively affects structural integrity of venous tissues. After the use of conserving solutions (Eurocollins and Scientific Research Institute of Traumatology and Orthopaedics) necrotized endotheliocytes were absent and endothelial lining was preserved in major part of the vein surface studied even following its deep freezing which supports the conclusion on the cryoprotective effect of the solutions.     

Diffusion and distribution of dimethyl sulphoxide in the isolated guinea-pig taenia coli

1. The diffusion of the cryoprotective non-electrolyte dimethyl sulphoxide (DMSO) in the isolated guinea-pig taenia coli at 37, 25 and 0 degrees C has been studied using [(35)S]DMSO.2. Within 1 hr after immersion at 37 degrees C in Krebs solution containing 20% (w/v) DMSO and trace amounts of [(35)S]DMSO, the non-electrolyte was distributed uniformly throughout a volume equivalent to the total initial water content of the muscle.3. The kinetics of efflux of [(35)S]DMSO from muscles at constant volume were analysed on the basis of two models: one incorporated radial diffusion in extracellular fluid with simultaneous permeation into the cells, the other involved only radial diffusion in homogeneous cylinders of tissue having no internal barriers to diffusion; the former was found to give a better representation of the efflux kinetics.4. If it was assumed that the rate of diffusion of DMSO in the extracellular space of taenia coli was the same as that in the bathing medium, the values of the extracellular space and the permeability of smooth muscle to DMSO, obtained from the analysis of the efflux kinetics, were 454 +/- 19 ml./kg and 2.36 +/- 0.05 x 10(-6) cm sec(-1) at 37 degrees C.5. The activation energy for the transfer of DMSO across the surface of the cell was estimated to be 6.0 kcal/mole at 37 degrees C, 6.6 kcal/mole at 25 degrees C and 11.6 kcal/mole at 0 degrees C, indicating either that the equivalent pore radius of the cells decreased with temperature or that the cell permeability represented the sum of two fluxes, one through the aqueous pores of the cell and the other through the lipid phase of the cell membrane, each with a different energy of activation.6. A net flux of water across the surface of the cells, superimposed on the efflux of DMSO, markedly affected the rate of diffusion of the non-electrolyte out of the whole tissue; however, it was considered that an analysis of the efflux kinetics was not possible under these conditions.7. These results provide a basis for methods which will be used to investigate the possibility of preserving tissue in unfrozen aqueous media at sub-zero temperatures.     

Cryopreservation of oocytes from pre-antral follicles

Cryobiology is a very important tool in reproductive biology. Research in this area focuses on the possibility of restoring fertility in women with reproductive problems or after cancer treatments. Another goal is to establish a genetic resource bank for endangered or commercially important animal species. Cryopreservation of oocytes from pre-antral follicles has been studied during the past decade. Procedures can be divided between the cryopreservation of either ovarian tissue or isolated follicles. Most studies describe a slow freezing/rapid thawing protocol to cryopreserve ovarian fragments. Histology shows that the follicles maintain their morphological integrity, and transplantation of ovarian tissue demonstrates that the follicles can restart their growth and eventually ovulate. Some research groups have obtained offspring using this procedure in mice and sheep. With regard to the cryopreservation of isolated follicles, the few studies reported in this area used the same freezing protocol, and some of them described follicular growth using in-vitro culture. The best result was obtained in mice, with animal birth after follicular cryopreservation and culture. However, additional studies are necessary for a better understanding of the events during follicular cryopreservation and to establish a standard protocol for ovarian transplantation or follicle culture.     

Cryopreservation does not affect proliferation and multipotency of murine neural precursor cells

Stem cell research offers unique opportunities for developing new medical therapies for devastating diseases and a new way to explore fundamental questions of biology. Establishing an efficient freezing protocol for neural precursor cells (NPCs) is of great importance for advances in cell-based therapies. We used fluorescence-activated cell sorter-based cell death/survival analysis and Western blot analysis of proliferation markers (proliferating cell nuclear antigen) and prosurvival proteins (Bcl-2) to study the effect of a variety of cryoprotective agents on fetal mouse forebrain NPCs. Neurospheres frozen at -70 degrees C or in liquid nitrogen in a rate-controlled manner and thawed after 5 days retained viability of 60%-70% measured 24 hours after thawing. However, 1 week after thawing, viability dropped to 50%-60%. Using a clonogenic sphere formation assay, we showed that recovery rate of frozen NPCs was approximately 26% and did not significantly differ between dimethyl sulfoxide (DMSO)- and glycerol-supplemented samples. Application of the caspase inhibitor zVAD-fmk during freezing or in the first week after thawing resulted in protection of cryopreserved neurospheres after thawing but not during the freezing process, indicating that apoptosis limits recovery of NPCs. Cell survival was not reduced in cells that were enzymatically separated before cryopreservation. Optimal protection of NPCs was achieved when 10% DMSO alone or in a combination with 10% fetal calf serum (FCS) was used. However, 10% glycerol alone was equally effective. Using these protocols, NPCs retained their multipotency and differentiated into both glial (GFAP-positive) and neuronal (Tuj1-positive) cells. Percentage of Tuj1-positive cells in 5% and 10% DMSO, in 10% DMSO + 10% FCS, and in 10% glycerol remained at the same level as before freezing and varied from 5%-7%. We conclude that cryopreservation (up to 1 month at -70 degrees C and up to 1 year in liquid nitrogen) does not markedly alter the rate of proliferation and multipotency of murine neural precursor cells.     

Immunological responsiveness of frozen-thawed human lymphocytes.

Mononuclear cells (10--20 X 10(6)) obtained from human peripheral blood by a standard Ficoll-Hypaque technique were suspended in RPMI 1640 media at 4 degrees C containing 10% foetal calf serum and 7-5% dimethyl sulphoxide (DMSO). Two-millilitre aliquots were cooled at -1 degree C/min in a Cryoson BV-4 programmed freezing system to -30 degrees C, then -5 degrees C/min to -80 degrees C and stored in liquid nitrogen vapor. On the day of testing, cell suspensions were thawed rapidly in a 37 degree C water bath. DMSO was diluted slowly out of the sample and cells resuspended in fresh RPMI 1640. It was found that frozen stored human lymphocytes (FSHL) demonstrated all the characteristics of fresh unfrozen cells. These included their ability to form spontaneous rosettes with sheep erythrocytes ('E' rosettes) and sheep erythrocyte--antibody--complement rosettes ('EAC' rosettes). The presence of surface immunoglobulins and Fc receptors were shown by membrane immunofluorescence to be comparable. In addition, the results show that FSHL respond to mitogens, specific antigens; act as both stimulators and responders in the mixed lymphocyte culture reaction; and exhibit cell-mediated lymphocytotoxicity following in vitro sensitization, or against antibody-coated target cells.