Cryopreservation,survival and function of intrastriatal fetal mesencephalic grafts in a rat model of Parkinson's disease

Summary In the present study we quantitatively assessed to what extent freeze-storage at liquid nitrogen temperature influences the survival and function of fetal mesencephalic grafts in the dopamine-depleted rat striatum. Ventral mesencephalic (VM) tissue was dissected from rat fetuses and stored overnight in a preservative medium at 4 °C (hibernation). It was grafted intrastriatally either as a fresh cell suspension or was frozen as tissue fragments or as a cell suspension after stepwise incubation in ascending concentrations of dimethyl-sulphoxide. Following a cryopreservation interval of 80 days in liquid nitrogen, the frozen samples were rapidly thawed, rinsed, and grafted. Cellular viabilities of graft cell suspensions, as assessed by ethidium bromide/acridine orange staining, were decreased from 90% in fresh tissue to 38-35% in frozen and thawed tissue. Amphetamine-induced turning behavior at 6 weeks post-grafting was significantly attenuated in hosts that had received fresh grafts or grafts that were frozen as tissue fragments. Tyrosine hydroxylase-(TH-) immunocytochemistry of recipient brains revealed significant decreases in TH-positive graft cell numbers in rats grafted with cryopreserved tissue (38–42% of fresh tissue). Moreover, the dye exclusion viability of thawed VM tissue was found to accurately predict the subsequent graft survival. There was no difference with respect to graft cell numbers between the two freezing methods employed, though block storage seems to be more simple from a practical point of view. The present study indicates that freezing in liquid nitrogen may be a feasible method for long-term storage of fetal neural tissue for grafting, although a marked decrease in graft survival and function of cryopreserved tissue must be taken into account.Present address: Dept. of Medical Physiology     

雪旺氏细胞从冷冻人胎周围神经的分离技术及其特性

本研究取１５～２０周人胎周围神经，放在冷冻的含ＤＭＥＭ（４０％）、ＤＭＳＯ（１０％）及小牛血清（５０％）中。细胞活性为９１％。雪旺氏细胞经过分离、培养、保持了活性。经３～６月液氮保存后，细胞活性为９４％，雪旺氏细胞在４℃下经２４小时，活性仍有９２％。从新鲜周围神经取得的和经冷冻保存的以及单个培养的雪旺氏细胞，在培养时生长缓慢，但在培养液中加牛脑垂体提取液后，细胞增殖加快。取自新鲜或冷冻的周围神经的雪旺氏细胞用Ｓ－１００蛋白抗体染色的特性相似。本文还报告了分离出的雪旺氏细胞在扫描及透射电镜下的形态特征。     

Cryopreservation of human granulocytes in liquid nitrogen.

Human granulocytes (PMNL) were successfully cryopreserved for up to 14 months. The PMNL (1-2 X 10(7)/ml) were stored in 2-ml ampoules in the gas phase of liquid nitrogen at a temperature between -160 degrees C and -196 degrees C using dimethylsulphoxide (DMSO 10%) as cryoprotectant. Morphology and phagocytic and bactericidal capacity were best preserved by adding fetal calf serum to the freezing mixture, by using an interrupted cooling process, by washing the thawed PMNL in fresh freeze-dried plasma, and centrifuging at 600 g for no more than two minutes. Careful post-thaw handling of the cells was an important factor in preserving function. These preliminary studies indicate that useful numbers of PMNL can be recovered in a functional state after storage for long periods in liquid nitrogen.     

Plaque-forming cell response of pokeweed mitogen stimulated frozen human lymphocytes

The plaque-forming cell (PFC) response of pokeweed mitogen (PWM) activated fresh or frozen human peripheral blood lymphocytes (PBL) was monitored by the protein A hemolytic plaque assay. Fresh PBL and PBL stored in nitrogen for 7 days to 8 years were tested. On the basis of our preliminary results we conclude that cryopreserved cells secrete the same amounts of immunoglobulin (Ig) as freshly prepared cells.     

The effects of freeze/thawing process on cryopreserved equine umbilical cord blood-derived mesenchymal stem cells

The use of mesenchymal stem cells (MSCs)-derived equine umbilical cord blood in cell grafts transplantation is advantageous; therefore, preservation of these cells is of utmost importance in repair therapies. To evaluate the viability ratio of the MSCs obtained from equine umbilical cord blood after cryopreservation, umbilical cord blood obtained from nine 46–48-week-old foals were purified for harvesting MCs. The purified cells were frozen from the first to tenth passages and stored in liquid nitrogen. After thawing, the cell viability was assessed through trypan blue staining procedure. The highest viability (>80%) ratio was observed with the cells derived from the first passage in 1 and 8?weeks after cryopreservation. However, the viability of cells was dependent on the passage used for cryopreservation. Results in this study demonstrated that equine umbilical cord blood stem cells could successfully be frozen and stored in liquid nitrogen for 8?weeks without significant change in the characterization of the cells cryopreserved as regards their viability, growth ability, and differentiation potential.     

Identification and reduction of cryoinjury in endothelial cells: a first step toward establishing a cell bank for vascular tissue engineering

We analyzed a cryopreservation protocol which improves long-term storage of endothelial cells (EC) for tissue engineering purposes. Human umbilical vein EC were frozen in a high-potassium solution containing 10% dimethyl sulfoxide using 3 different cooling rates. After a storage time in liquid nitrogen of 1, 4, or 12 months, samples were thawed and compared to fresh cells in terms of growth rates, anti-inflammatory, and anticoagulant functions. Independent of cooling rate and storage time, the retrieval after cryopreservation ranged between 60% and 80%. However, viability of the cells cryopreserved at 10 degrees C/min decreased significantly from 78 +/- 5% to 64 +/-3% with storage. Storage time of 4 months resulted in a decreased cell multiplication factor over 4 and 12 days in culture. The lag phases returned to normal in the next passage. Thawed cells showed increased metabolic activity, reduced expression of thrombomodulin, and unchanged basal expression of adhesion molecules. However, the tumor necrosis factor-induced expression of adhesion molecules was significantly increased after long-term storage. This effect was partially compensated after expansion of the cells, whereas the prostacyclin release increased. Expansion of cryopreserved/thawed EC resulted in highly proliferative cells with antithrombotic properties and a capacity for inflammatory reactions, which makes them suitable for vascular tissue engineering.     

Routine freezing of red blood cells for transfusion in Western Australia.

Experience with preservation of red cells for transfusion since 1971 has shown that refrigeration with liquid nitrogen is simple and reliable. It allows the use of a concentration of glycerol (20%) which can reasonably be removed by batch-washing if automated washing is not possible. It is possible to transport cells frozen in liquid nitrogen over long distances. This appears the method of choice for preserving rare blood, and once adopted for this purpose it is logical to use it for storage of red cells for other patients. Red cells so stored are similar in many respects to fresh cells, and more than 600 units have been transfused since 1971 without any adverse reaction.     

Cryopreservation of antibody-coupled red cells for use in immunoassays

Red cells, trypsin-treated to render them more agglutinable and coupled with antiglobulin reagents, may be preserved by droplet freezing in liquid nitrogen. A 2% cell suspension in 0.45% w/v sodium chloride, 5% w/v sucrose and 10% w/v dextran 40 was used. After thawing the frozen pellets in phosphate-buffered saline at 40 degrees C, more than 80% cell recovery was obtained. Sheep and ox red cells preserved in this way were as satisfactory in antiglobulin and in reverse passive haemagglutination tests as unfrozen indicator red cells. Trypsin-treated human red cells coupled with anti-IgE could likewise be frozen and on reconstitution used to assay IgE in human serum. Reconstituted ox red cells were slightly less efficient in rosetting than cells which had not been frozen.     

T-cell subset analysis of cryopreserved human peripheral blood mononuclear cells

A criticism of current techniques for monitoring changes in T-cell subset numbers over extended periods in individuals with disease states in which such changes might provide insight is the fact that serial samples taken are usually analysed fresh and therefore not in the same assay. To try to overcome this problem we have stored peripheral blood mononuclear cells frozen in liquid nitrogen and thence examined their ability to form sheep red blood cell (E) rosettes and to label with OK monoclonal antibodies. Results obtained show that cell viabilities following freezing and T-cell subset analysis of E-rosette positive cells are no different when fresh or frozen and subsequently thawed peripheral blood mononuclear cells are used.     

Isolation of DNA from archival Papanicolaou stained cytological smears using a simple salting-out procedure

DNA from archival Papanicolaou stained and unstained cytological smears was successfully isolated using a simple, rapid and inexpensive salting-out procedure. The quality of DNA was controlled by polymerase chain reaction (PCR) amplification of segments of the human β-globin, human β-actin and human papillomavirus L1 genes. Only negligible differences in amplification efficiency were observed between DNA isolated from stained and unstained smears. The salting-out procedure is a more rapid method for the isolation of DNA than phenol-chloroform extraction and may be used in instances where fresh or cryopreserved clinical specimens are not available.     

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.     

A Cryopreservation Method of Human Peripheral Blood Mononuclear Cells for Efficient Production of Dendritic Cells

The establishment of a cryopreservation method for unstimulated fresh peripheral blood mononuclear cells (PBMC) with nearly 100% viability would greatly contribute to the conduct of various immunological experiments. The cells most sensitive to freezing and thawing procedure seem to be dendritic cells (DC) and their precursors, which are of the most potent antigen-presenting cells. The authors investigated and established a method of cryopreserving fresh PBMC from which DC were recovered and differentiated efficiently by using recombinant (r) GM-CSF and rIL-4. PBMC frozen in the presence of 12% dimethylsulfoxide and 25–30% fetal calf serum recovered DC as efficiently as freshly obtained PBMC. Established DC could also be cryopreserved in the presence of 12% DMSO with their viability maintained at more than 90%. The 12% DMSO freezing solutions were superior to both the 10% DMSO solution and the previously reported DC freezing medium (2 m or 15.4% DMSO). The DC obtained from the cryopreserved PBMC expressed HLA-DR, HLA-DQ, CD80 and CD86 antigens, and stimulated allogenic PBMC to an extent almost identical to that obtained from fresh PBMC. These findings indicate that the conditioned medium utilized here enables safe cryopreservation of DC and DC precursors in PBMC.     

Feasibility of vitrification as a storage method for tissue-engineered blood vessels

It is well established that, in multicellular systems, conventional cryopreservation results in damaging ice formation, both in the cells and in the surrounding extracellular matrix. As an alternative to conventional cryopreservation, we performed a feasibility study using vitrification (ice-free cryopreservation) to cryopreserve tissue-engineered blood vessels. Fresh, frozen, and vitrified tissue-engineered blood vessels were compared using histological methods, cellular viability, and mechanical properties. Cryosubstitution methods were used to determine the location of ice in conventionally cryopreserved engineered vessels. Ice formation was negligible (0.0 +/- 0.0% of vessel area) in the vitrified specimens, and extensive (68.3 +/- 4.5% of vessel area) in the extracellular matrix of frozen specimens. The metabolic assay and TUNEL staining results indicated that vitrified tissue had similar viability to fresh controls. The contractility results for vitrified samples were >82.7% of fresh controls and, in marked contrast, the results for frozen samples were only 10.7% of fresh controls (p < 0.001). Passive mechanical testing revealed enhanced tissue strength after both freezing and vitrification. Vitrification is a feasible storage method for tissue-engineered blood vessel constructs, and their successful storage brings these constructs one step closer to clinical utility.     

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.     

Viability and Recovery of Peripheral Blood Mononuclear Cells Cryopreserved for up to 12 Years in a Multicenter Study

The Multicenter AIDS Cohort Study (MACS), an ongoing prospective study of the natural history of human immunodeficiency virus (HIV), has stored biologic specimens, including peripheral blood mononuclear cells (PBMC), from 5,622 participants for up to 12 years. The purpose of the present analysis was to evaluate the quality of the PBMC in the MACS repository in order to test the validity and feasibility of nested retrospective studies and to guide the planning of future repositories. PBMC were collected from MACS participants at four centers at 6-month intervals from 1984 to 1995, cryopreserved, and transported to a central repository for storage. A total of 596 of these specimens were subsequently tested for viability and used to evaluate cell function, to conduct immunophenotype analysis, or to isolate HIV. Simple linear regression models were applied to evaluate trends in recovery and viability over time and by center. Results indicated that from a nominal 10⁷ cells cryopreserved per vial at all four centers, the median number of viable cells recovered was at least 5 × 10⁶ (50% of the number stored) and the median viability was at least 90%. Results suggested that cryopreserved cells can be stored for at least 12 years with no general tendency toward cell loss over time. Furthermore, there were no statistically significant changes in the percent cell viability according to the length of time frozen, regardless of HIV serostatus or the level of CD4⁺ lymphocytes. Storing 10⁷ PBMC per vial yields sufficient viable cells for phenotypic and/or functional analysis. Results from the MACS provide the basis for the planning of future repositories for use by investigators with similar research goals.     

Effect of cryopreservation on measurement of cytomegalovirus-specific cellular immune responses in HIV-infected patients

To determine the feasibility of cytomegalovirus (CMV)-specific cell-mediated immunity (CMI) studies using cryopreserved cells, we compared lymphocyte proliferation assays (LPA), responder cell frequency (RCF), interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) production using fresh and cryopreserved peripheral blood mononuclear cells (PBMCs) from 53 HIV-infected patients and 15 uninfected controls. Qualitative CMV LPA results were concordant in >/=84% of the specimens from either HIV-infected patients or controls. Proliferation-based RCF, IL-2, and IFN-gamma comparisons showed that cryopreservation reduces the number of CMV-specific responders and decreases cytokine secretion, without changing the rank order of the results (p <.01). In contrast, the number of flow cytometry-enumerated IFN-gamma-producing CD4+ cells was not significantly changed by cryopreservation. In HIV-infected patients, the differences between fresh and frozen cell assays were not influenced by CD4 cell numbers or HIV viral load. These data indicate that cryopreserved cells are suitable for longitudinal studies of the CMV-specific immune response in HIV-infected patients and uninfected controls.     

Flow-cytometric screening of platelet antibodies with previously frozen cells

In this study the flow-cytometric crossmatch results were compared between fresh cells and cells processed by various cryopreservation and storage methods. Platelets from healthy donors were incubated with 12 sera containing platelet reactive antibodies as well as with 62 control sera from blood donors. Direct comparisons were made between fresh platelets and platelets after freezing at -28 degrees C, -40 degrees C and -80 degrees C and in liquid nitrogen, using 6% DMSO as cryoprotectant. In addition, the effects of using controlled-rate freezing were evaluated. Finally we evaluated the application of the cryoprotectant ThromboSol. The best results were obtained after cryopreservation of the platelets with ThromboSol at -80 degrees C with controlled cooling rates. Using ThromboSol cryopreserved platelets, the sensitivity for the detection of incompatible platelets was 100% and the specificity was 97.1%, using the previous results obtained with flow-cytometry, MAIPA and LCT as a reference. CONCLUSION: Platelets can be frozen using ThromboSol as the cryoprotectant, with controlled rate freezing and storage at -80 degrees C for the screening of platelet antibodies and for flow-cytometric crossmatch procedures. This system yields a reproducible and logistically simple method for platelet crossmatching that yields results superior to fresh cells and can be easily incorporated into standard clinical laboratory practices.     

Cryopreserved versus freshly isolated lymphocytes in human biomonitoring: endogenous and induced DNA damage, antioxidant status and repair capability

Lymphocytes are routinely used in human biomonitoring to assess the potential toxic and cytoprotective effects of diet on both DNA damage and repair and, by implication, health. Logistically, samples may require to be cryopreserved and stored. How this affects cells used in human biomonitoring is often not considered. In this study we have evaluated the influence of cryopreservation on endogenous and induced DNA strand breakage, altered bases (oxidized purines, oxidized pyrimidines and misincorporated uracil), antioxidant capacity and DNA repair capability in human peripheral blood lymphocytes. Neither isolation nor freezing increased DNA strand breakage above endogenous levels found in freshly isolated human lymphocytes. Oxidized bases (both pyrimidines and purines) and misincorporated uracil, were similar for fresh and frozen lymphocytes. Fresh and frozen lymphocytes responded almost identically to hydrogen peroxide. Quercetin-mediated cytoprotection against hydrogen peroxide-induced strand breakage was maintained in cryopreserved lymphocytes after short-term (24 h) and longer term (2 months) storage compared with freshly isolated and treated cells. Hydrogen peroxide-induced DNA strand breakage was repaired in fresh lymphocytes. Cryopreserved lymphocytes were unable to repair oxidant-induced DNA strand breaks. Frozen human lymphocytes can therefore be successfully used for most aspects of DNA damage biomonitoring, but not for repair.     

Evaluation of a method for cryogenic storage of cytological specimens

A technique for long-term storage of cytological specimens at — 70°C was evaluated with a range of nongynaecological cytology specimens. The effects of frozen storage on the cellularity, morphology, ultrastructure, and reactivity to cytochemical and immunochemical stains were investigated. These parameters were compared in preparations made from specimens processed conventionally and after frozen storage. No significant deterioration in the quality of subsequent preparations was apparent after storage. The method is technically simple and has proven successful for storing a range of specimens, including serous effusions, urine samples, and fine-needle aspirates. This technique allows storage of material until initial microscopic evaluation is complete. If required, the relevant adjuvant procedure, such as cell blocking, may then be carried out using the stored material. Wastage of cytological material on unnecessary preparations is minimised, and material may be stored over years for research or confirmation procedures.     

Survivability of rabbit amniotic fluid-derived mesenchymal stem cells post slow-freezing or vitrification

This work aimed to evaluate the effect of two distinct cryopreservation procedures - conventional slow-freezing and vitrification, on survivability and mesenchymal marker expression stability of rabbit amniotic fluid-derived mesenchymal stem cells (rAF-MSCs). Cells at passage 2 were slowly frozen, using 10% of dimethylsulfoxide, or vitrified, using 40% of ethylene glycol, 0.5 M sucrose and 18% Ficoll 70. After three months storage in liquid nitrogen, viability, chromosomal stability, ultrastructure, surface and intracellular marker expression and differentiation potential of cells were evaluated immediately post-thawing/warming and after additional culture for 48–72 h. Our results showed decreased (P ≤ 0.05) viability of cells post-thawing/warming. However, after additional culture, the viability was similar to those in fresh counterparts in both cryopreserved groups. Increase (P ≤ 0.05) in the population doubling time of vitrified cells was observed, while doubling time of slow-frozen cells remained similar to non-cryopreserved cells. No changes in karyotype (chromosomal numbers) were observed in frozen/vitrified AF-MSCs, and histological staining confirmed similar differentiation potential of fresh and frozen/vitrified cells. Analysis of mesenchymal marker expression by qPCR showed that both cryopreservation approaches significantly affected expression of CD73 and CD90 surface markers. These changes were not detected using flow cytometry. In summary, the conventional slow-freezing and vitrification are reliable and effective approaches for the cryopreservation of rabbit AF-MSCs. Nevertheless, our study confirmed affected expression of some mesenchymal markers following cryopreservation.