A comparison of a sucrose-based solution with other preservation media for cold storage of isolated hepatocytes

A sucrose-based solution has been compared with other preservation solutions (University of Wisconsin (UW) solution and Marshall's citrate solution, with Dulbecco's medium as control) during hypothermic preservation of isolated rat hepatocytes for up to 72 h. Studies on the stability of liver cells at low temperature by exclusion of trypan blue dye and morphological appearance were conducted. During storage beyond 24 h, there was a clear difference between cells stored in Dulbecco's medium and Marshall's citrate and those stored in sucrose-based solution and UW solution, with the former storage groups showing many cells developing large membrane "blebs" and the latter storage groups maintaining a more typical morphology and developing only small membrane protrusions. Dye exclusion was higher in sucrose-based solution (48 h, 75 +/- 7%; 72 h, 65 +/- 6%) and UW solution (48 h, 72 +/- 5%; 72 h, 63 +/- 4%) than in Marshall's citrate (48 h, 31 +/- 5%; 72 h, 10 +/- 1%) and Dulbecco's medium (48 h, 8 +/- 2%; 72 h, 5 +/- 1%). These data suggest that sucrose-based solution should be investigated further as a less complex alternative solution for storage of isolated hepatocytes.     

Endothelial cell preservation at hypothermic to normothermic conditions using clinical and experimental organ preservation solutions

Introduction

Endothelial barrier function is pivotal for the outcome of organ transplantation. Since hypothermic preservation (gold standard) is associated with cold-induced endothelial damage, endothelial barrier function may benefit from organ preservation at warmer temperatures. We therefore assessed endothelial barrier integrity and viability as function of preservation temperature and perfusion solution, and hypothesized that endothelial cell preservation at subnormothermic conditions using metabolism-supporting solutions constitute optimal preservation conditions.Methods: Human umbilical vein endothelial cells (HUVEC) were preserved at 4–37 °C for up to 20 h using Ringer's lactate, histidine–tryptophan–ketoglutarate solution, University of Wisconsin (UW) solution, Polysol, or endothelial cell growth medium (ECGM). Following preservation, the monolayer integrity, metabolic capacity, and ATP content were determined as positive parameters of endothelial cell viability. As negative parameters, apoptosis, necrosis, and cell activation were assayed. A viability index was devised on the basis of these parameters.Results: HUVEC viability and barrier integrity was compromised at 4 °C regardless of the preservation solution. At temperatures above 20 °C, the cells' metabolic demands outweighed the preservation solutions' supporting capacity. Only UW maintained HUVEC viability up to 20 °C. Despite high intracellular ATP content, none of the solutions were capable of sufficiently preserving HUVEC above 20 °C except for ECGM.Conclusion: Optimal HUVEC preservation is achieved with UW up to 20 °C. Only ECGM maintains HUVEC viability at temperatures above 20 °C.     

Glycine prevention of cold ischemic injury in isolated hepatocytes

Isolated hepatocytes suspended in a liver preservation solution (University of Wisconsin (UW) solution) and exposed to cold (5 degrees C) ischemia lose viability (LDH release) after 3 (76.5 +/- 2.6% extracellular LDH) and 4 days (90.3 +/- 5.7% extracellular LDH) storage when rewarmed (37 degrees C) in Krebs-Henseleit buffer. However, if 3 mM glycine is added to Krebs-Henseleit buffer the loss of LDH on rewarming was suppressed (% LDH = 24.4 +/- 2.2% and 33.2 +/- 3.0%, at 3 and 4 days, respectively). The protection by glycine could also be obtained by storing the hepatocytes in the UW solution containing 15 mM glycine and rewarming in the absence of glycine in Krebs-Henseleit buffer. There did not appear to be a relationship between the protection by glycine and glutathione concentration of the hepatocytes as shown by the lack of effect of a glutathione synthetase inhibitor (butathionine sulfoximine) on the protective effects of glycine. Other amino acids did not provide protection to hepatocytes exposed to cold ischemia. The mechanism of action of glycine is not known, but this compound may be important in improving cold storage of livers for transplantation.     

Subzero nonfreezing cryopresevation of rat hearts using antifreeze protein I and antifreeze protein III

The purpose of the present study was to evaluate whether AFPs protect the heart from freezing and improve survival and viability in subzero cryopreservation. Hearts were subject to 5 preservation protocols; University of Wisconsin solution (UW) at 4 degrees C, UW at -1.3 degrees C without nucleation, UW at -1.3 degrees C with nucleation, UW AFP I (15 mg/cm(3)) at -1.3 degrees C with nucleation, and in UW AFP III (15 mg/cm(3)) at -1.3 degrees C with nucleation. Hearts were preserved for 24, 28, and 32 h, rewarmed and connected to the working isolated perfusion system. Data [heart rate (HR), coronary flow (CF), and developed pressure (dP)] was collected 30 and 60 min after reperfusion. Hearts preserved at -1.3 degrees C without AFPs froze, while hearts preserved with AFP did not freeze when nucleation was initiated and survived. Survival and dP of hearts preserved for 24h at -1.3 degrees C using AFP III was better than those preserved at 4 degrees C, (dP; 1.4 vs. 0.8, p<0.05). Four of six hearts and six of six hearts died when preserved at 4 degrees C for 28 and 32 h, respectively, all of the hearts that were preserved at -1.3 degrees C with or without AFPs survived after 28 h (n=18) and 32 h (n=18). CF was higher in UW -1.3 degrees C group without attempted nucleation than in AFP I and AFP III groups after 28 and 32 h (3.4 vs. 1.7, p<0.05, and 3.4 vs. 1.7, p<0.05, respectively). In conclusion, AFPs were found to protect the heart from freezing and improve survival and dP (AFP III) in prolonged subzero preservation.     

Effect of phospholipase A2 inhibitors on the release of arachidonic acid and cell viability in cold-stored hepatocytes

We investigated the effect of phospholipase A(2) (PLA(2)) inhibitors on PLA(2) activity and cell viability in cold-stored rat hepatocytes. The cells were radiolabeled with [(3)H] arachidonic acid (AA) and cold stored in the University of Wisconsin (UW) solution containing various PLA(2) inhibitors. PLA(2) activity was determined by measuring the total free (cellular + supernatant) AA by thin-layer chromatography after inhibiting reacylation of free AA with inhibitors of energy production (carbonyl cyanide m-chlorophenylhydrazone + iodoacetate). Aristolochic acid, chlorpromazine, and quinacrine in the UW solution showed a significant inhibitory effect throughout 48 h cold storage but only at relatively high concentration. PLA(2) activity was also suppressed (58% of control) by trifluoperazine (50 microM), but its effect was limited to only 24 h. In contrast, pretreatment of the cells prior to hypothermic preservation with trifluoperazine (10 to 100 microM) suppressed PLA(2) activity during 48 h storage. Inclusion of calmodulin antagonist W-7 did not affect PLA(2) activity. Thus, the inhibitory activity of these agents appears unrelated to Ca-calmodulin-phospholipid interaction but to have an inhibitory effect on PLA(2) activity. To study the effects of PLA(2) inhibitors on cell viability, lactate dehydrogenase (LDH) release was measured in the presence or absence of inhibitors upon rewarming cold-stored cells in Krebs-Henseleit buffer for 2 h at 37 degrees C. None of the inhibitors tested improved cell viability after 48 h storage. Thus, although PLA(2) inhibitors blocked PLA(2) activity, there was no suppression of LDH release. PLA(2) may play a minor role in preservation/reperfusion injury to cold-stored hepatocytes.     

Skim milk powder used as a non-permeable cryoprotectant reduces oxidative and DNA damage in cryopreserved zebrafish sperm

Cryoprotectants play a vital role in the cryopreservation process, protecting biological samples from freezing damage. Here, we evaluate the effects of the combination and interaction of different extenders with permeable and non-permeable cryoprotectants, on the cryopreservation of Danio rerio sperm, analyzing the effects of cryopreservation through a broad approach to variables. Two extenders were used, Hank's balanced salt solution (HBSS) and Ginsburg's solution. Eight cryoprotective solutions (CS) were used: CS1 (HBSS + Me₂SO 8%), CS2 (HBSS + Methanol 8%), CS3 (HBSS + Me₂SO 8% + Skim milk powder 15%), CS4 (HBSS + Methanol 8% + Skim milk powder 15%), CS5 (Ginsburg + Me₂SO 8%), CS6 (Ginsburg + Methanol 8%), CS7 (Ginsburg + Me₂SO 8% + Skim milk powder 15%) and CS8 (Ginsburg + Methanol 8% + Skim milk powder 15%). The samples were cryopreserved in cryovials for 20 min on dry ice, stored in liquid nitrogen, thawed at 38 °C for 10 s, and analyzed. In addition to increasing viability, we show that powdered milk also allows for better preservation of the membrane and normal cell morphology, and protects the sperm cells from DNA damage and oxidative stress caused by cryopreservation.     

Hypothermic preservation of hepatocytes : I. Role of cell swelling

Hepatocytes from isolated rat livers were hypothermically incubated (5 degrees C) in an oxygenated environment with continuous shaking (to simulate organ perfusion preservation). The incubation solution was either a tissue culture medium (L-15), an organ preservation perfusate (UW gluconate), or a simple cold-storage solution used for organ preservation (UW lactobionate). Hepatocyte viability was assessed from the release of lactate dehydrogenase (LDH) into the incubation medium. Cell swelling (due to the uptake of water) was also measured. Within 24 hr, hepatocytes hypothermically stored in each of the three incubation solutions became swollen (30 to 40% water gain) and lost a significant amount of LDH (as much as 60%). The addition of polyethylene glycol (PEG; relative molecular mass 8000; 5 g%) to the solutions suppressed cell swelling and allowed the incubated hepatocytes to remain relatively well preserved (30% LDH release) for as long as 120 hr. Adding either dextran (relative molecular mass 10,000 to 78,000; 5 g%) or saccharides (100 mmol/liter) instead of PEG neither prevented cell swelling nor prevented the cells from dying. The results of this study suggest (i) there is a direct correlation (r = 0.873) between hypothermia-induced cell swelling and cell death (i.e., the suppression of cell swelling prevents cell death); (ii) the mechanism by which PEG prevents cell swelling (and thus maintains cell viability) is not related to the osmotic or oncotic properties of the molecule but instead is apparently related to some unknown interaction between PEG and the cell, an interaction that provides stability during hypothermic incubation; and (iii) hypothermia-induced cell swelling must be prevented if isolated hepatocytes are to be used as a model for studying the mechanism by which cell damage occurs during hypothermic organ preservation. By eliminating cell death due to cell swelling, the biochemical mechanisms of cell death can be studied.     

Cryopreservation at −80°C of Agaricus blazei on rice grains

The preservation of Agaricus blazei is generally done by mycelial subculturing, but this technique may cause genetic degenerations. Despite this, there is not an efficient protocol established to preserve this fungus and cryopreservation could be an alternative. This study aimed to evaluate two freezing protocols for cryopreservation at −80°C of A. blazei strains. Five fungus strains grown on rice grains with husk and were transferred to glycerol (10%) in cryovials. Next, the cryovials were submitted to two freezing temperature protocols: (1) cryopreservation starting at 25°C, then at 8°C for 30 min and kept at −80°C; (2) cryopreservation starting at 25°C, then 8°C for 30 min, −196°C for 15 min and kept at −80°C. After 1 year of cryopreservation, the cryovials were thawed in a water bath at 30°C for 15 min and transferred to malt extract agar medium. It was concluded that the one-year cryopreservation process of A. blazei, grown on rice grains and cryopreserved at −80°C in glycerol 10%, is viable. The slow freezing, from 8 to −80°C, is effective whereas the fast freezing, from 8 to −196°C and then to −80°C, is ineffective. The different genetic characteristics among the strains of this fungus do not interfere in the cryopreservation process.     

Viability,attachment efficiency,and xenobiotic metabolizing enzyme activities are well maintained in EDTA isolated rat liver parenchymal cells after hypothermic preservation for up to 3 days in University of Wisconsin solution

Summary Rat liver parenchymal cells were isolated by EDTA perfusion and were subsequently purified by Percoll centrifugation. The freshly isolated liver cells had a mean viability of 95% as judged by trypan blue exclusion. Isolated liver parenchymal cells were then stored at 0°C for up to 1 wk in University of Wisconsin solution (UW). During this hypothermic preservation, the viability was only slightly reduced to 92% after 1 d and to 85% after 3 d at 0°C. Thereafter, the viability decreased rapidly. After cold storage for up to 3 d, it was possible to use the parenchymal liver cells either in short-term suspension or in cell culture. The attachment efficiency in cell culture was the same for freshly isolated liver cells (84%) and after 2 d cold preservation (81%). The cytochrome P450 content and the enzyme activities of soluble expoxide hydrolase, UDP-glucuronosyl transferase, phenol sulfotransferase, and glutathione S-transferase were not significantly different between freshly isolated cells and cells after 3 d of hypothermic preservation. Furthermore, freshly isolated and intact liver cells stored for 3 d were used in the cell-mediated Salmonella mutagenicity test as a metabolizing system. Both fresh and stored liver parenchymal cells metabolized benzo(a)pyrene, 2-aminoanthracene, and cyclophosphamide to their ultimate mutagens. Thus, it was clearly demonstrated that EDTA-isolated liver parenchymal cells retain their xenobiotic metabolizing capacity after short-term hypothermic preservation for up to several days and, therefore, may help to maximize the usefulness of rarely available liver parenchymal cells such as those from humans and help to reduce the number of experimental animals required for pharmacological and toxicologicalin vitro investigations.     

Methodology for processing mastectomy and cryopreservation of breast cancer tissue in a resource- poor setting: A pilot study

BackgroundThere is scarcity of breast cancer tissues derived from women of African origin available for patient - derived xenograft and organoid models.ObjectiveWe aim to create a versatile protocol for processing mastectomy and cryopreservation of breast cancer tissue.MethodologyAn immediate collection of breast cancer tissue from mastectomy was bathed in 4 °C HBSS and immediately transferred to 4 °C RPMI1640 containing HEPES, 10% FBS, Streptomycin and Penicillin. Tissues were processed over ice yielding nine samples of cold ischemic time (20–45 min) stored at 3 min interval. Cut samples were transferred into cryovials containing 4 °C cryoprotectant agent (90% FBS +10% Me₂SO) before snap -freezing in liquid Nitrogen vapour and final short-term storage in −80 °C Freezer. The histomorphology, tissue and molecular viability were assessed.ResultsThe cold ischemic times had no detrimental effect to the nine samples despite being processed in a resource poor setting, hence providing a reproducible and reliable protocol.     

Optimizing amniotic membrane tissue banking protocols for ophthalmic use

Amniotic membrane (AM) due to its anti-inflammatory, anti-scarring and anti-angiogenic properties is used as corneal and wound grafts. When developing AM tissue banks, cell viability, membrane morphology and genomic stability should be preserved following cryopreservation. To analyze the changes rendered to the AM during the process of cryopreservation by comparing different combinations of standard cryopreservation media; fetal bovine serum (FBS), dimethyl sulfoxide (DMSO), Dulbecco’s modified eagle’s medium (DMEM) and glycerol at ?80 °C and at ?196 °C for a period of 6 weeks and at 4 °C in 70 % alcohol for 6 weeks. Following informed consent, placentae of healthy term pregnancies delivered by elective Cesarean section were collected and AM separated into 5 × 5 cm size sections and under sterile conditions stored in 9:1 DMSO:FBS and 1:1 DMEM:Glycerol at ?196 and ?80 °C for 6 weeks. Similar sections were also stored at 4 °C in 70 % alcohol for 6 weeks. After storage periods following were assessed; AM epithelial cell viability by trypan blue vital stain, epithelial cell proliferation capacity by cell doubling time, membrane morphology by haematoxylin and eosin (H&E) stain and genomic stability by conventional G-banded karyotyping. Human amniotic epithelial cells were cultured in DMEM and 10 % FBS in humidified atmosphere of 5 % carbon dioxide at 37 °C and were characterized using RT-PCR for Octamer-binding protein 4 (Oct-4) and glucose-6-phosphate dehydrogenase (G6PD) genes. All the above parameters were also assessed in fresh AM. AM obtained from 4 term placentae. Mean cell count and mean cell doubling times in days respectively; for fresh AM 3.8 × 10⁶; 1.59, after 6 weeks in DMSO:FBS at ?196 °C 3.0 × 10⁶; 2.38 and at ?80 °C 2.1 × 10⁶; 1.60, in DMEM:Glycerol at ?196 °C 3.6 × 10⁶; 2.33 at ?80 °C 23 × 10⁶; 1.66 and at 4 °C 3.3 × 10⁶; 2.14. Histology analysis of the fresh AM showed an intact epithelial monolayer, thick basement membrane (BM) and avascular stromal matrix. Amniotic membranes stored at ?196 °C showed morphology similar to fresh AM in both preservation media and AM stored at ?80 °C showed disruption of the stromal matrix. At 4 °C the epithelial monolayer showed flattening. Fresh AM karyotype was 46XX. Analyzable spreads for karyotype were not obtained from stored AMs. Human amniotic epithelial cells were positive for both Oct-4 and G6PD genes. AM is best preserved at ?196 °C either in 1:9 DMSO:FBS or 1:1 DMEM:Glycerol. In both conditions cell viability and membrane integrity were shown to be preserved up to 6 weeks. Since analyzable chromosome spreads from cell cultures were not obtained, genomic stability could not be assessed.     

Long-term,large scale cryopreservation of insect cells at −80 °C

Standard tissue culture methods advise freezing cells in small aliquots (≤1 × 10⁷ cells in 1 mL), and storing in liquid nitrogen. This is inconvenient for laboratories culturing large quantities of insect cells for recombinant baculovirus expression, owing to the length of time taken to produce large scale cultures from small aliquots of cells. Liquid nitrogen storage requires use of specialized cryovials, personal protective equipment and oxygen monitoring systems. This paper describes the long-term, large scale cryopreservation of 8 × 10⁸ insect cells at −80 °C, using standard 50 mL conical tubes to contain a 40 mL cell suspension. Sf9, Sf21 and High 5 cells were recovered with a viability > 90 % after storage for one year under these conditions, which compared favorably with the viability of cells stored in liquid nitrogen for the same length of time. Addition of green fluorescent protein encoding baculovirus demonstrated that cells were “expression ready” immediately post thaw. Our method enables large scale cultures to be recovered rapidly from stocks cryopreserved at −80 °C, thus avoiding the inconvenience, hazards and expense associated with liquid nitrogen.

Electronic supplementary material

The online version of this article (doi:10.1007/s10616-014-9781-5) contains supplementary material, which is available to authorized users.     

Ischemic preconditioning in a rodent hepatocyte model of liver hypothermic preservation injury

Ischemic preconditioning (IPC) is a phenomenon of protection in various tissues from normothermic ischemic injury by previous exposure to short cycles of ischemia-reperfusion. The ability of IPC to protect hepatocytes from a model of hypothermic transplant preservation injury was tested in this study. Rat hepatocytes were subjected to 30min of warm ischemia (37 degrees C) followed by 24 or 48h of hypothermic (4 degrees C) storage in UW solution and subsequent re-oxygenation at normothermia for 1h. Studies were performed with untreated control cells and cells treated with IPC (10min anoxia followed by 10min re-oxygenation, 1 cycle). Hepatocytes exposed to IPC prior to warm ischemia released significantly less LDH and had higher ATP concentrations, relative to untreated ischemic hepatocytes. IPC significantly reduced LDH release after 24h of cold storage before reperfusion and after 48h of cold storage and after 60min of warm re-oxygenation, relative to the corresponding untreated hepatocytes. ATP levels were also significantly higher when IPC was used prior to the warm and cold ischemia-re-oxygenation protocols. In parallel studies, IPC increased new protein synthesis and lactate after cold storage and reperfusion compared to untreated cells but no differences in the patterns of protein banding were detected on electrophoresis between the groups. In conclusion, IPC significantly improves hepatocyte viability and energy metabolism in a model of hypothermic preservation injury preceded by normothermic ischemia. These protective effects on viability may be related to enhanced protein and ATP synthesis at reperfusion.     

A sterile leakproof plastic vial for cell cryopreservation in liquid nitrogen: Application to parasitic protozoa

Polyethylene bulbs derived from sterile transfer pipets were evaluated as leak-proof vials for cryopreservation of parasitic protozoa. The bulbs were easily frozen and stored in conventional liquid nitrogen refrigeration equipment and were less expensive and safer to handle than plastic screw-capped cryovials. The easily testable heat seal of the polyethylene bulb never failed during longterm immersion in liquid nitrogen, and dispensing of thawed material was simple and safe. Good survival of L. donovani (80%), T. rhodesiense (95%), and C. fasciculata (95%) was obtained after cryopreservation in polyethylene bulbs or plastic screw-capped vials. The polyethylene bulb was considered an excellent container for the safe cryopreservation of pathogens and other biological material.     

Energy status of isolated hepatocytes after long-term cold storage in various types of media

Using of isolated hepatocytes for investigation of the effects of hypothermia, it has been demonstrated that sucrose-base solution provides of maintenance of the energetic parameters (level of ATP, glucose synthesis, rate of gluconeogenesis) within 48 hrs of storage at 4 degrees C. It efficiency was compared with effect on the energetic status of isolated hepatocytes widely used preservation solution--solution of University Wisconsin (UW). After long-term of cold storage of isolated hepatocytes (72 hrs) at 4 degrees C in both solutions, it has been shown sharp decrease of ATP level (on two time). Viability of the liver cells (in both cases) was practically without change.     

p NMR Studies of Rat Liver Cold Preservation with Histidine-Buffered Lactobionate Solution

The efficiency of a preservation medium, histidine-buffered lactobionate solution (HBLS), was determined by measuring post-ischemic recoveries of ATP and intracellular pH under Krebs-Henseleit buffer (KHB) perfusion. We used NMR spectroscopy to study the effect of 24-h cold ischemia, followed by 4°C then 37°C reperfusion on the isolated rat liver. Three media were compared: University of Wisconsin solution (UW-lactobionate); Bretschneider's solution (HTK); HBLS and HBLS supplemented with 2 mM Gly and 2 mM Cys (HBLSg2) or with 10 mM Gly and 2 mM Cys (HBLSg10). All values were compared to control values measured during pre-ischemic cold perfusion with KHB (ATP = 8.60 ± 0.6 μmol/g of dry weigh and pH_in = 7.41 ± 0.05). The main result from ³¹p NMR data concerned ATP recovery during cold reperfusion, which was significantly higher in the HBLS group (112 ± 10%) as compared to the UW and HTK groups (around 66%). The presence of glycine decreased ATP recovery (88 ± 8% in HBLSg2, 79 ± 15% in HBLSg10). Higher values of recovered pH_in were observed in livers stored in histidine buffered solutions (around 7.30) as compared to UW (around 7.20); histidine was by ¹³C NMR proved to accumulate in the liver cells, thus ensuring a good buffering capacity. The thermal transition induced a decrease in both ATP level and pH_in in all groups. This might be the result of a stimulation of the carbohydrate metabolism (as demonstrated by ¹³C NMR) especially when glycine was present in the storage solution.     

Extracellular calcium protects cultured rat hepatocytes from injury caused by hypothermic preservation

Effects of various preservation solutions were compared in an experimental hypothermic preservation model using cultured rat hepatocytes. Hepatocytes prepared by the collagenase perfusion method were cultured for 48 hr, then the medium in each culture dish was exchanged for various preservation solutions, and the dishes were hypothermically (0-2 degrees C) stored in a refrigerator for 12-72 hr. After the preservation period, the hepatocytes were cultured again at 37 degrees C for 2 hr. Hepatocytes' viability after 18-hr preservation and reculture was greater when they were preserved in "intracellular" rather than "extracellular" solutions. Even with Euro-Collins solution (intracellular solution), hepatocyte viability decreased to approximately 20% after 24-hr preservation, and an increase in the cellular lipid peroxide content was observed. However, when this solution contained a submillimolar concentration of calcium, lipid peroxidation was significantly suppressed and hepatocyte viability was dramatically improved. Vitamin E was almost equally effective and a marked synergistic effect was observed with calcium. Calcium was found to be capable of maintaining the cellular glutathione level during cold storage, which seems to suppress lipid peroxidation and consequently improve hepatocyte survival.     

Response of squash to ethylene and chilling

Freshly harvested but already cured Acorn squash (Cucurbita pepo cv. Acorn) were stored for 4 wk in storage environments of 20, 15, 10 , 5 and o ^oC, with or without 5 μl 1-1^-1 ethylene. Ethylene had no significant effect on overall acceptability, weight loss, dry matter, and ion leakage, but did enhance respiration at 15 and 20 ^oC. Temperature had a significant effect on weight loss, dry matter and overall acceptability. Squash stored at 15 and 20 ^oC had better flavour and texture than those stored at 0–10 ^oC, when cooked. Quality attributes such as flavour and texture may be more useful than physiological parameters in detecting insipient chilling injury in squash.     

A comparative study of freezing single cells and spheroids: Towards a new model system for optimizing freezing protocols for cryobanking of human tumours

Cryopreservation of human tumour cells and tissue is a valuable tool for retrospective analysis and for the transport and handling of biopsy material. Tumour tissue consists of different cell types, which have different optimal freezing conditions, and extracellular matrix. A well-defined and authentic model system is required for developing new freezing protocols and media. This work describes the use of L929 and PC-3 spheroids as new model systems for freezing human tumours. Cell suspension and spheroids were frozen in different vessels (1 ml cryovials and a special, cryo-compatible 30 × 25 μl multi well plate) at slow rate (1 °C/min). Freezing media were combinations of culture or tumour transport medium (Liforlab^®) with the cryoprotective agents, Me₂SO, trehalose and modified starch. We also present a new method of evaluating the viability of three dimensional multicellular systems to compare thawed spheroids objectively. Best viability (70%) of L929 spheroids occurred with a combination of Liforlab^® and starch hydrolysis product. The best cryopreservation results for spheroids were found with extracellular cryoprotectants, while optimum viability of single cells was achieved with Me₂SO.