Embryonic development of the sea urchin after low-temperature preservation

The sea urchin embryos were cooled to -196 degrees by two-step freezing with the use of 1-1.5 M dimethyl sulfoxide as a cryoprotectant. The embryos were equilibrated with the cryoprotectant for 20-30 min at 0 +/- 2 degrees. At -7 degrees ice crystallization was induced and the embryos were cooled to -38-42 degrees at a rate of 6-8 degrees /min. The embryos were then transferred into liquid nitrogen. The embryos were thawed in a water bath at 19 degrees. No less than 90% of the embryos frozen at the stages of blastula, gastrula, or pluteus were capable of recovery and normal development. The length of cryopreservation did not affect the survival of the embryos.     

Cryopreservation of human endothelial cells for vascular tissue engineering

To investigate the influence of cryopreservation on endothelial cell growth, morphology, and function human umbilical vein endothelial cells (HUVECs) were frozen following a standard protocol. Cell suspensions were exposed to 10% dimethyl sulfoxide in a high-potassium solution, cooled to -80 degrees C at 1 degrees C/min and stored in liquid nitrogen for 7-36 days. Samples were thawed in a 37 degrees C water bath and the cryoprotectant was removed by serial dilution. The growth of cell suspensions was assayed by culturing 7300 cells/cm2 for 3-5 days in order to determine the cell multiplication factor. Fresh and cryopreserved/thawed cells were analyzed for their growth, and their anti-inflammatory and anti-coagulant function by using cellular ELISA. Cryopreservation resulted in a retrieval of 66 +/- 5% and a viability of 79 +/- 3%. Cryopreserved/thawed and fresh cells showed identical doubling times and identical cell counts in the confluent monolayers. However, the lag phase of thawed HUVECs was approximately 36 h longer, resulting in significant differences in the cell multiplication factor at 3 and 5 days after seeding. After expansion to a sufficient cell count the lag phases were identical. Fresh and cryopreserved/thawed cells showed comparable anti-inflammatory and anti-coagulant activity, as judged by the basal and TNF-induced VCAM-1, ICAM-1, E-selectin, and thrombomodulin expression. Cryopreserved/thawed and recultivated endothelial cells are suitable for endothelialization of autologous allograft veins. Such tissue-engineered grafts will offer the necessary clinical safety for those patients who lack autologous material.     

Corneal cryopreservation with dextran.

Different methods of corneal cryopreservation have been introduced, those employing intracellular cryoprotectants such as Me2SO or glycerol being the most widely favored. We investigated the influence of several freeze-thaw trauma variables on the survival of porcine endothelial monolayers when employing the extracellular cryoprotective agent dextran. We first examined the effects of various dextran concentrations and then, having ascertained the optimal concentration, further investigated the influence of fetal calf serum (FCS) concentration in the cryopreservation medium, the cooling rate, the thawing temperature, and the length of the preincubation in the freezing medium prior to cryopreservation. The numerical densities of endothelial cells were determined at dissection in hypoosmotic balanced salt solution and after organ culture by staining with alizarin red S and trypan blue. Morphological evaluation was not performed directly after thawing but after a subsequent organ culture at 37 degrees C to detect latent cell damage after freeze-thaw trauma. Our data revealed that corneas cryopreserved in minimal essential medium containing 10% dextran but lacking FCS, preincubated for 3 h, frozen at a cooling rate of 1 degrees C/min, and thawed at 37 degrees C incurred the lowest cell losses (22.4%, SD +/- 3.8). We conclude that dextran is an effective cryoprotectant for freezing of porcine corneas. However, variations between species in the results of cryopreservation require further investigation of an in vivo animal model and studies with human corneas before its clinical use can be recommended.     

The potential of an equimolar combination of propane-1,2-diol and glycerol as a vitrification solution for corneas

Corneas must first be equilibrated with multimolar concentrations of cryoprotectants if the formation of ice during cryopreservation is to be avoided by vitrification at practicable cooling rates. Rabbit corneas were exposed to equimolar mixtures of the cryoprotectants propane-1,2-diol and glycerol in a Hepes-buffered Ringer's solution containing glutathione, adenosine, 5 mmol/liter sodium bicarbonate, and 6% w/v bovine serum albumin. Endothelial function was assessed by monitoring its ability to control stromal hydration during perfusion of the endothelial surface at 34 degrees C for 6 h. Endothelial morphology was observed by specular microscopy during perfusion and by scanning electron microscopy after perfusion. Endothelial pump activity and structural integrity of the endothelial layer were demonstrated after 20 min exposure at 4 degrees C to a total concentration of 1.4 mol/liter cryoprotectant (i.e., 0.7 mol/liter propane-1,2-diol + 0.7 mol/liter glycerol). Exposure to 2.0 and 3.4 mol/liter cryoprotectant for 20 min at 4 degrees and -5 degrees C, respectively, resulted in initial endothelial damage; but this repaired and a functioning endothelial pump was subsequently demonstrated. Although exposure to 4.1 mol/liter cryoprotectant for 10 min at -10 degrees C caused irreparable damage to 2/4 corneas, reduced dilution temperatures together with increased dilution time allowed exposure to 4.8 and 5.5 mol/liter cryoprotectant with retention of endothelial pump activity. Exposure to 6.1 mol/liter cryoprotectant for 10 min at -15 degrees C caused endothelial damage which was not mitigated by the presence of 2.5% w/v chondroitin sulfate. Endothelial function may be improved by further modification of addition and dilution protocols or by exposure to the cryoprotectants at lower temperatures.     

Expansion and cryopreservation of porcine and human corneal endothelial cells

Impairment of the corneal endothelium causes blindness that afflicts millions worldwide and constitutes the most often cited indication for corneal transplants. The scarcity of donor corneas has prompted the alternative use of tissue-engineered grafts which requires the ex vivo expansion and cryopreservation of corneal endothelial cells. The aims of this study are to culture and identify the conditions that will yield viable and functional corneal endothelial cells after cryopreservation. Previously, using human umbilical vein endothelial cells (HUVECs), we employed a systematic approach to optimize the post-thaw recovery of cells with high membrane integrity and functionality. Here, we investigated whether improved protocols for HUVECs translate to the cryopreservation of corneal endothelial cells, despite the differences in function and embryonic origin of these cell types. First, we isolated endothelial cells from pig corneas and then applied an interrupted slow cooling protocol in the presence of dimethyl sulfoxide (Me₂SO), with or without hydroxyethyl starch (HES). Next, we isolated and expanded endothelial cells from human corneas and applied the best protocol verified using porcine cells. We found that slow cooling at 1 °C/min in the presence of 5% Me₂SO and 6% HES, followed by rapid thawing after liquid nitrogen storage, yields membrane-intact cells that could form monolayers expressing the tight junction marker ZO-1 and cytoskeleton F-actin, and could form tubes in reconstituted basement membrane matrix. Thus, we show that a cryopreservation protocol optimized for HUVECs can be applied successfully to corneal endothelial cells, and this could provide a means to address the need for off-the-shelf cryopreserved cells for corneal tissue engineering and regenerative medicine.     

New approach to improving endothelial preservation in cryopreserved arterial substitutes

The endothelial loss provoked by the methods of vascular cryopreservation used at most human vessel banks is one of the main factors leading to the failure of grafting procedures performed using cryopreserved vessel substitutes. This study evaluates the effects of the storage temperature and thawing protocol on the endothelial cell loss suffered by cryopreserved vessels, and optimises the thawing temperature and protocol for cryopreserving arterial grafts in terms of that producing least endothelial loss. Segments of the common iliac artery of the minipig (n = 20) were frozen at a temperature reduction rate of 1 degrees C/min in a biological freezer. After storing the arterial fragments for 30 days, study groups were established according to the storage temperature (-80, -145 or -196 degrees C) and subsequent thawing procedure (slow or rapid thawing). Fresh vessel segments served as the control group. Once thawed, the specimens were examined by light, transmission, and scanning electron microscopy. The covered endothelial surface was determined by image analysis. Data for the different groups were compared by one way ANOVA. When cryopreservation at each of the storage temperatures was followed by slow thawing, the endothelial cells showed improved morphological features and viability over those of specimens subjected to rapid thawing. Rapidly thawed endothelial cells showed irreversible ultrastructural damage such as mitochondrial dilation and rupture, reticular fragmentation, and peripheral nuclear condensation. In contrast, slow thawing gave rise to changes compatible with reversible damage in a large proportion of the endothelial cells: general swelling, reticular dilation, mitochondrial swelling, and nuclear chromatin condensation. Gradually thawed cryopreserved arteries showed a lower proportion of damaged cells identified by the TUNEL method compared to the corresponding rapidly thawed specimens (p < 0.05, for all temperatures). In all the groups in which vessels underwent rapid thawing (except at -145 degrees C), significant differences (p < 0.05) in endothelial cover values were recorded with respect to control groups. Storage of cryopreserved vessels at -80 degrees C followed by rapid thawing led to greatest endothelial cell loss (61.36+/-9.06% covered endothelial surface), while a temperature of -145 degrees C followed by slow thawing was best at preserving the endothelium of the vessel wall (89.38+/-16.67% surface cover). In conclusion, storage at a temperature of -145 degrees C in nitrogen vapour followed by gradual automated thawing seems to be the best way of preserving the endothelial surface of the arterial cryograft. This method gives rise to best endothelial cell viability and cover values, with obvious benefits for subsequent grafting.     

The cryobiology of rat and human dendritic cells: preservation and destruction of membrane integrity by freezing

Dendritic cells (DCs) are now regarded as specialized leucocytes with distinctive morphological and functional characteristics as accessory or stimulator cells for many lymphocyte responses. While knowledge of the response of other leucocytes (e.g., lymphocytes, macrophages, and granulocytes) to freezing and thawing has been established for some years, an understanding of the cryobiological properties of DCs has not, hitherto, been determined specifically. Such information is important both for establishing procedures for the long-term storage of these cells for use in immunological procedures and for defining freezing conditions that might selectively kill DCs in attempts to modulate the immunogenicity of transplantable tissues during cryopreservation. Preparations of rat and human spleen cells enriched for DCs were frozen to -60 degrees C at one of six cooling rates (0.3, 1.5, 10, 20, 70, or 150 degrees C/min) using a procedure that was established for pancreatic islets with 2 M dimethyl sulfoxide (Me2SO) as the cryoprotectant. Following storage at -196 degrees C the survival of thawed cells was assessed by evaluating both the numbers of cells recovered after the complete process and the membrane integrity of the recovered cells using a supravital fluorescent probe assay. Survival profiles for DCs showed a dependence upon cooling rate similar to other lymphoid cells but DCs were more sensitive to freezing injury than either lymphocytes or macrophages: Optimum survival (75% recovery of numbers and 57% membrane integrity) of rat DCs was achieved by slow cooling (0.3 degrees C/min). Optimal recovery of human DCs was significantly higher (83% recovery of numbers and 72% membrane integrity) after cooling at either 0.3 or 1.5 degrees C/min. The viable yield of DCs from both species declined abruptly as cooling rate was increased, with less than 10% survival after cooling at 20 degrees C/min and negligible survival after cooling at 70 degrees C/min or greater. Analysis of variance of the survival data showed that the response of DCs to freezing and thawing was significantly different (P less than 0.005) from that of either lymphocytes or macrophages, thus providing additional evidence that DCs are distinct from other leucocytes, especially macrophages. This study defines conditions that either will provide effective cryopreservation of DCs for immunological purposes or are most likely to bring about their inactivation in cryobiological approaches to modulating tissue immunogenicity.     

Corneal tolerance of vitrifiable concentrations of glycerol.

Equilibration of corneas with sufficiently high concentrations of cryoprotectants to inhibit potentially damaging ice formation during cryopreservation has not yet been achieved. This study examined the effects on the structure and function of rabbit corneal endothelium of the low toxicity cryoprotectant glycerol. Corneas were exposed to concentrations ranging from 2.0 to 6.8 M glycerol in a Hepes-buffered Ringer's solution containing glutathione, adenosine, 5 mM sodium bicarbonate and 6% w/v bovine serum albumin. Endothelial function was assessed by monitoring corneal thickness during perfusion of the endothelial surface at 34 degrees C for 6 h. Endothelial structure was observed using specular microscopy during perfusion and scanning electron microscopy after perfusion. Corneas tolerated exposure to 2.0 and 3.4 M glycerol for 20 min at 4 and -5 degrees C, respectively. Tolerance of 4.8 M glycerol for 10 min at -10 degrees C was improved by decreasing the dilution temperatures. Ten-minute exposure to 6.1 and 6.8 M glycerol was tolerated at -15 degrees C. In all cases corneas initially showed signs of damage but endothelial function was regained following structural repair. Corneas exposed to 6.8 M glycerol and cooled below the glass transition temperature were nonfunctional after warming. Ice formation during warming was believed to be the cause of injury.     

Defining the optimal cryoprotectant and concentration for cryopreservation of limbal stem cells

Limbal stem cell (LSC) deficiency causes progressive loss of vision but may be treated by transplant of autologous LSCs. Cryopreservation has the potential to indefinitely extend the lifespan of LSCs allowing re-transplant in case of graft failure. In this study, we aimed to identify the optimal cryoprotectant and cryoprotectant concentration for LSC cultures. Suspension cultures derived from cadaveric corneoscleral rims were cooled to 4 °C with Me₂SO, propylene glycol or ethylene glycol at a concentration of 5%, 10% or 15%. Cell tolerance was measured in terms of membrane integrity, colony-forming efficiency and alamarBlue^® reduction. Increasing cryoprotectant concentration above 5% reduced membrane integrity, metabolism and colony-forming efficiency. Cryoprotectant choice did not significantly influence these characteristics. Cells demonstrating Side Population were maintained after cryopreservation with 5% propylene glycol in vapour phase liquid nitrogen for 1 week, indicating that cryopreservation of LSCs with relatively low cryoprotectant concentration (5%) has promise in low-temperature eye banking.     

Drug metabolism and viability studies in cryopreserved rat hepatocytes

Rat hepatocytes were cryopreserved optimally by freezing them at 1 degrees C/min to -80 degrees C in cryoprotectant medium containing either 20% (v/v) dimethylsulfoxide (Me2SO) and 25% (v/v) fetal calf serum in Leibowitz L15 medium (Me2SO cryoprotectant) or 25% (v/v) vitrification solution (containing Me2SO, acetamide, propylene glycol and polyethylene glycol) in Leibowitz L15 medium (VS25). The VS25 solution was superior for maintaining viability during short-term storage (24-48 hr) but was slightly toxic during longer storage periods (7 days). Although thawed cells were 40-50% viable on ice after cryopreservation, their viability fell rapidly during incubation in suspension at 37 degrees C. This decline in viability occurred more rapidly after freezing in Me2SO cryoprotectant than in VS25 and was associated with extensive intracellular damage and cell swelling. The loss in viability at 37 degrees C does not appear to be due to ice-crystal damage as it occurred in cells stored at -10 degrees C (above the freezing point of the cryoprotectants) and it may be due to temperature/osmotic shock. Both cryoprotectant media were equally efficient at preserving enzyme activities in the hepatocytes over 7 days at -80 degrees C. Cytochrome P450 and reduced glutathione content and the activities of the microsomal enzymes responsible for aminopyrine N-demethylation and epoxide hydrolysis were well maintained over 7 days storage. In contrast, the cytosolic enzymes glutathione-S-transferase and glutathione reductase were markedly labile during cryopreservation. Cytosolic enzymes may be more susceptible to ice-crystal damage, whereas the microsomal membrane may protect the enzymes which are embedded in it.     

Transplantation of cryopreserved human corneas in a xenograft model

An ideal model to test methods of corneal storage for transplantation would simulate the environment of the grafted human cornea and predict the success of clinical corneal transplants (human to human). In this study, we tested such a model, the corneal xenograft (human to cat). Nine pairs of human corneas were transplanted into both eyes of nine recipient cats. One cornea of each pair was cryopreserved at -196 degrees C in 2.5 M dimethyl sulfoxide while the other was stored in preservative medium at 4 degrees C (control) for 6 +/- 2 (mean +/- SD) days before transplantation. One week after transplantation, the cats were euthanized and the eyes were examined. Three of the grafts (all cryopreserved) were clinical failures and showed no survival of donor corneal endothelial cells on scanning electron microscopy. The remaining six pairs of grafts were examined with a specular microscope and showed endothelial cell losses of 48 +/- 16% in cryopreserved and 8 +/- 16% in control corneas (p < 0.05). This survival is similar to survival in an earlier corneal perfusion model. The nine cryopreserved grafts were thicker than the control grafts, had fewer surviving keratocytes in the central stroma, and had more apoptotic central keratocytes (TUNEL assay). This failure rate in cryopreserved corneas clearly shows that this technique of cryopreservation was not adequate for clinical use. The corneal xenograft model can be used to study cellular survival and apoptosis in vivo after preservation as well as to test new methods of corneal preservation before initiating clinical trials.     

Cryopreservation of cornea: a low cooling rate improves functional survival of endothelium after freezing and thawing

AIM: To investigate the influence of low cooling rates on endothelial function and morphology of corneas frozen with propane-1,2-diol (PROH). METHODS: Rabbit corneas, mounted on support rings, were exposed to 1.4mol/l (10% v/v) PROH, seeded to initiate freezing, and cooled at 0.2 or 1 degrees C/min to -80 degrees C. Corneas were frozen immersed in liquid or suspended in air. After being held overnight in liquid nitrogen, corneas were warmed at 1 or 20 degrees C/min. After stepwise removal of the cryoprotectant, the ability of the endothelium actively to control corneal hydration was monitored during normothermic perfusion. Morphology was assessed after staining with trypan blue and alizarin red S, and by specular microscopy during perfusion. RESULTS: Functional survival was achieved only after slow cooling (0.2 degrees C/min) with the cornea immersed in the cryoprotectant medium, and rapid warming (20 degrees C/min). These conditions also gave the best morphology after freezing and thawing. CONCLUSION: Cooling rates lower than those typically applied to cornea improved functional survival of the endothelium. This result is in accord with previous observations showing the benefit of low cooling rates for cell monolayers [CryoLetters 17 (1996) 213-218].     

Serum- and albumin-free cryopreservation of endothelial monolayers with a new solution

Cryopreservation is the only long-term storage option for the storage of vessels and vascular constructs. However, endothelial barrier function is almost completely lost after cryopreservation in most established cryopreservation solutions. We here aimed to improve endothelial function after cryopreservation using the 2D-model of porcine aortic endothelial cell monolayers.?The monolayers were cryopreserved in cell culture medium or cold storage solutions based on the 4°C vascular preservation solution TiProtec^®, all supplemented with 10% DMSO, using different temperature gradients. After short-term storage at ?80°C, monolayers were rapidly thawed and re-cultured in cell culture medium.?Thawing after cryopreservation in cell culture medium caused both immediate and delayed cell death, resulting in 11 ± 5% living cells after 24 h of re-culture. After cryopreservation in TiProtec and chloride-poor modifications thereof, the proportion of adherent viable cells was markedly increased compared to cryopreservation in cell culture medium (TiProtec: 38 ± 11%, modified TiProtec solutions ≥ 50%). Using these solutions, cells cryopreserved in a sub-confluent state were able to proliferate during re-culture. Mitochondrial fragmentation was observed in all solutions, but was partially reversible after cryopreservation in TiProtec and almost completely reversible in modified solutions within 3 h of re-culture. The superior protection of TiProtec and its modifications was apparent at all temperature gradients; however, best results were achieved with a cooling rate of ?1°C/min.?In conclusion, the use of TiProtec or modifications thereof as base solution for cryopreservation greatly improved cryopreservation results for endothelial monolayers in terms of survival and of monolayer and mitochondrial integrity.     

Survival of frozen mouse embryos after rapid thawing from -196 degrees C.

The effect of the rate of rewarming on the survival of 8-cell mouse embryos and blastocysts was examined. The samples were slowly cooled (0.3--0.6 degrees C/min) in 1.5 M-DMSO to temperatures between -10 and -80 degrees C before direct transfer to liquid nitrogen (-196 degrees C). Embryos survived rapid thawing (275--500 degrees C/min) only when slow cooling was terminated at relatively high subzero temperatures (-10 to -50 degrees C). The highest levels of survival in vitro of rapidly thawed 8-cell embryos were obtained after transfer to -196 degrees C from -35 and -40 degrees C (72 to 88%) and of rapidly thawed blastocysts after transfer from -25 to -50 degrees C (69 to 74%). By contrast, for embryos to survive slow thawing (8 to 20 degrees C/min) slow cooling to lower subzero temperatures (-60 degrees C and below) was required before transfer to -196 degrees C. The results indicate that embryos transferred to -196 degrees C from high subzero temperatures contain sufficient intracellular ice to damage them during slow warming but to permit survival after rapid warming. Survival of embryos after rapid dilution of DMSO at room temperature was similar to that after slow (stepwise) dilution at 0 degrees C. There was no difference between the viability of rapidly and slowly thawed embryos after transfer to pseudopregnant foster mothers. It is concluded that the behaviour of mammalian embryos subjected to the stresses of freezing and thawing is similar to that of other mammalian cells. A simpler and quicker method for the preservation of mouse embryos is described.     

Corneal tolerance of vitrifiable concentrations of propane-1,2-diol

The merit of corneal cryopreservation by vitrification as opposed to conventional freezing is the avoidance of ice damage which is believed to disrupt the integrity of the corneal endothelium resulting in loss of corneal transparency. The cornea must be equilibrated with high concentrations of cryoprotectant in order to achieve vitrification at practicable cooling rates. In an earlier study, corneas were exposed to 3.4 mol/liter propane-1,2-diol (Rich and Armitage (1990) Cryobiology 27, 42-54). The present study exposed rabbit corneas to concentrations of propane-1,2-diol between 3.4 and 5.4 mol/liter in a Hepes-buffered Ringer's solution containing glutathione, adenosine, 5 mmol/liter sodium bicarbonate, 6% (w/v) bovine serum albumin, and 2.5% (w/v) dextran sulfate. Dextran sulfate was as effective as chondroitin sulfate at improving endothelial tolerance of 3.4 mol/liter propane-1,2-diol. This beneficial effect may be linked to the polyanionic nature of these molecules. Corneas exposed to 5.4 mol/liter propane-1,2-diol were cooled in liquid nitrogen vapor at a temperature of -140 degrees C for 2 h. Warming was achieved by direct transfer to a dilution solution at -10 degrees C. Endothelial function was assessed by monitoring corneal thickness during perfusion of the endothelial surface at 34 degrees C for 6 h. Endothelial structure was observed by specular microscopy during the perfusion and by scanning electron microscopy after perfusion. Corneas tolerated exposure to 3.4 mol/liter propane-1,2-diol for 20 min at 0 degrees C and to 4.1 mol/liter for 10 min at -10 degrees C. Exposure to 4.8 and 5.4 mol/liter for 10 min at -10 degrees C caused endothelial damage, although a degree of endothelial function was retained. Function following exposure to 5.4 mol/liter was improved by reducing the temperature of exposure to -15 degrees C. Corneas cooled after exposure to 5.4 mol/liter propane-1,2-diol for 10 min at -15 degrees C apparently vitrified, but devitrified on warming. The corneas swelled to such an extent during perfusion that the endothelium could not be viewed by specular microscopy, subsequent scanning electron microscopy showed a severely disrupted endothelium.     

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.     

Cryopreservation of Echinococcus multilocularis metacestodes and subsequent proliferation in rodents (Meriones)

Four isolates of larval Echinococcus multilocularis originating from Switzerland (CH/1, CH/6 and CH/22) and Alaska (A/1) were used to prepare crude homogenate or small tissue fragments (STF) in Eagle's Minimal Essential Medium with Earle's salts (EMEM/A), or 0.2 g tissue blocks (TB) which were suspended in the same medium. After addition of dimethylsulfoxide or glycerol in final concentrations of 5% and 10% (v/v), respectively, aliquots of 1.0 ml, containing either 0.1 ml crude homogenate or STF, or one block of 0.2 g, were kept in cryotubes for 30 min at +2-4 degrees C (precooling phase), cooled subsequently to lower temperatures following a two-step or three-step schedule and finally plunged into liquid nitrogen (-196 degrees C). After storage for one week the samples were rapidly thawed at +37 degrees C for approximately 3 min, washed in fresh EMEM/A (37 degrees C) and transferred into the peritoneal cavity of Meriones for viability testing. As judged by histological examinations and metacestode weights of each 24 Meriones infected with cryopreserved homogenate, STF or TB, respectively, 46%, 87% or 100% contained viable, proliferating parasites. The best proliferation rate occurred when 10% glycerol was used as cryoprotectant and after precooling a three-step freezing schedule was employed (30 min at -28 degrees C, 30 min at -80 degrees C, transfer to liquid nitrogen). Cooling rates were determined as 0.7, 1.0 and 1.7 degrees C min-1 for the precooling phase, step 1 and step 2, respectively, and estimated as 65 degrees C min-1 for step 3. These results demonstrate that metacestodes of E. multilocularis can be successfully maintained by cryopreservation without losing their proliferative capacity in the intermediate host.     

Cryopreservation of vascular endothelial cells as isolated cells and as monolayers

This paper reports the cryopreservation of an immortalized human endothelial cell line (ECV304), either as a single cell suspension or as a confluent layer on microcarrier beads. Cell suspensions were exposed to 10% w/w dimethyl sulfoxide in a high-potassium solution (CPTes) at 0 degrees C. The cells were then cooled to -60 degrees C at controlled rates between 0.3 and 500 degrees C/min and stored below -180 degrees C. Samples were thawed in a 37 degrees C water bath and the cryoprotectant was removed by serial dilution at 22 degrees C over 6 min. The recovery of cell suspensions was assayed by culturing aliquots in 24-well plates for 7-9 days and counting the number of colonies that contained >25 cells. Maximum survival was 45-50% at cooling rates of 0.3, 1.0, and 10 degrees C/min, but decreased to 20% at 50 degrees C/min and to <1% at 500 degrees C/min. Biosilon microcarrier beads were used for the attached cells. Confluent beads were cryopreserved by exactly the same technique and cell function was assayed by measuring active amino acid (leucine) transport at 37 degrees C. Control, untreated confluent beads gave approximately 73% of control uptake and negative controls (frozen without cryoprotectant) gave approximately 4% uptake. The cells attached to beads showed percentage uptakes that were numerically similar to the survival of cells in suspension at cooling rates between 10 and 500 degrees C/min, but at lower cooling rates the recovery of attached cells increased to 70% at 1 degrees C/min and to 85% at 0.3 degrees C/min. These results indicate a marked difference in the effect of cooling rate on ECV304 cells depending upon attachment.     

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.     

Comparison of three methods for human corneal cryopreservation that utilize dimethyl sulfoxide.

We compared endothelial cell survival in human corneas after cryopreservation by three methods that utilize dimethyl sulfoxide. Twenty-eight human cadaver corneas were cryopreserved by one of three methods, stored briefly over liquid nitrogen, thawed, cultured at 37 degrees C for 3 days, and fixed for scanning electron microscopy. Seventeen control corneas underwent identical cryoprotectant immersion and culture protocols but were not frozen. Endothelial photographs taken after 1 and 3 days of culture were analyzed. Endothelial cell losses in cryopreserved corneas by Methods 1, 2, and 3, respectively, were 36, 22, and 10% after 1 day of culture and 57, 36, and 27% after 3 days of culture. Cryopreservation by Method 3 had less cell loss than Methods 1 or 2 (P<0.02) but greater cell loss than the control corneas for Method 3 (P<0.001). No loss of cells occurred in the control corneas for Methods 1 and 3 but substantial cell loss (26%) occurred in the control corneas for Method 2. Polymegethism and pleomorphism of the endothelial cells were seen in the corneas that lost cells. The endothelial cell loss of 10% seen after 1 day of culture in human corneas cryopreserved by Method 3 is similar to the loss that occurs during organ culture storage as currently used clinically and therefore would be acceptable for clinical use. After 3 days of culture, however, the cell loss had increased significantly to 27%. This additional decrease in cell number that occurs in culture may represent latent cryodamage and must be understood and overcome in vivo before the technique can be used clinically.