Mechanical Freezer Research Articles

Cryopreservation of Platelets Isolated with the IBM 2997 Blood Cell Separator: A Rapid and Simplified Approach

The equivalent of 5-7 units of platelets, isolated from a single donor with the IBM Blood Cell Processor 2997 using a dual stage separation chamber, was frozen with the cryoprotectant dimethylsulfoxide (DMSO). The DMSO-saline solution was added directly to the platelets, and the platelets were frozen in a polyvinyl chloride plastic bag by storage in a -80 °C mechanical freezer. Washing the thawed platelets with a phosphate-buffered sodium chloride-dextrose solution, pH of 5.0, removed about 95% of the DMSO. In vitro freeze-thaw-wash recovery was 80%, and in vivo 51Cr platelet recovery was 31%. Platelet dense body granules were well maintained after freezing, thawing, and washing. This is a safe and effective method of platelet cryopreservation which can be performed in less time than other currently used methods.

Cryopreservation of human platelets isolated by discontinuous-flow centrifugation using the Haemonetics Model 30 Blood Processor.

Platelets were isolated from normal volunteers by discontinuous-flow centrifugation using the Haemonetics Model 30 Blood Processor. The numerical equivalent of about five single units of platelets collected at each pheresis were frozen together in a -80 C mechanical freezer with a 6% final concentration of dimethylsulfoxide (DMSO) as the cryoprotectant. Platelet freeze-thaw-wash recovery in vitro was about 80 per cent and the platelet recovery value depended upon the method used to enumerate the platelets. The 51Cr survival values in vivo were about 50 per cent less than those in fresh platelets. These values were not significantly different from those seen when platelets were isolated from single units of blood by differential serial centrifugation. Transfusion of two and one-half units of freeze-preserved platelets provided an increase in the recipient's circulating platelet count comparable with that from one unit of fresh platelets. The hemostatic effectiveness of freeze-preserved platelets isolated by discontinuous-flow centrifugation has not yet been studied.

Factors affecting the stability of cryogenically preserved granulocytes

Human granulocytes free of other cell types were obtained by counterflow centrifugation, cryogenically preserved, and studied for stability and function after thawing. Isolation of granulocytes by counterflow centrifugation was optimal at reduced temperatures (4–10 °C) in phosphate-buffered saline (or Ca 2+-free buffers) at pH 7.1. A stabilizing protein, or HES was required. Routinely, 1.2% human or bovine serum albumin was used. Hyperosmolar (310 m0sm) buffers and post isolation handling in ice water baths was optimal for cryogenic preservation. Addition of DMSO at 22 °C produced transient shrinkage initially which depended on the rate of addition, concentration, and temperature. Within 10–15 min granulocytes returned to volume, but continued to swell, equilibrating for 1 hr at 20% larger volume. Ethidium uptake gradually increased. After 24 hr, extreme swelling, lysis, and ethidium uptake was observed at the highest concentration (10%) of DMSO. DMSO-induced swelling was prevented with HES. Granulocytes (30 × 10 6 − 50 × 10 6) were frozen in 2.0-ml volumes in plastic tubes. The combination of 5% DMSO, 6% HES, 4% albumin, 0.056 M glucose in NormosolR at pH 7.1 produced the best yields. Granulocytes were first cooled to 4 °C, then to −80 °C (approx rate 4 °C per min) in a mechanical freezer and finally stored in liquid nitrogen. Storage varied from days to months. Granulocytes were thawed at 42 °C by manually twirling the freezing tubes and they were subsequently maintained in ice water. They were diluted 3:1 dropwise with a room temperature solution of 7% HES, 1.2% albumin, and 0.026 M glucose in Normosol. Particle ingestion tests were conducted by incubation at room temperature for forty minutes with yeast or zymosan opsonized with autologous serum. Particles ingested were counted by microfluorimetry after two washings at 150g. Granulocytes could not be cryogenically preserved in plasma or serum. Heating or prefreezing of serum was ineffective, but dialysis or addition of EDTA overcame the destructive effect of serum. Neither treatment was an improvement over the standard freeze procedure using buffered albumin and cryoprotective components. β-mercaptoethanol added to the freezing medium caused the production of a single homogeneous population of osmotically inert, nonviable, ethidium-reactive granulocytes. This suggests that osmoregulation by granulocyte membranes is a critical requirement for cryopreservation. Preservation efficiency is species dependent, increasing in the order of human, baboon, guinea pig, and dog. Dog granulocytes can be stored for at least 8 months in liquid nitrogen with small loss of cells and functionality. The present efficiency of preservation of human granulocytes for 3–4 weeks of liquid nitrogen storage is 90–100% morphological and 40% functional recovery. Attempts to increase stability of thawed granulocytes with other additions to our current procedure have so far proved fruitless. These have consisted of inosine, adenine, pyruvate, gluconate, vitamin C, β-mercaptoethanol, para-phenylmethyl-sulfonylfluoride, and mannitol.

Cryopreservation of Human Lymphocytes: A Brief Review and Evaluation of an Automated Liquid Nitrogen Freezer

Successful cryopreservation of human lymphocytes has been previously described. Cryopreserved lymphocytes are useful for a variety of in vitro immunologic studies. This study was performed to determine the applicability and/or advantages of using a programmable freezing system, and compares glycerol versus dimethyl sulfoxide (DMSO) at varying concentrations on post-thaw viability, E-rosetting and immunoglobulin fluorescence. Prefreeze T and B lymphocyte percentages were determined. Cells were then frozen in varying concentrations of glycerol and DMSO. Optimum cryoprotectant type and concentration was determined. Lymphocytes from seven individuals were frozen by the batch method in a mechanical freezer and with the automated liquid nitrogen injection system. Data on post-thaw T and B percentages and viability revealed 10% DMSO and liquid nitrogen control freezing method at 1 C/minute as the best conditions for lymphocyte preservation as reflected by post-thaw in vitro testing.

INFLUENCE OF LIQUID NITROGEN, LIQUID CARBON DIOXIDE AND MECHANICAL FREEZING ON SENSORY PROPERTIES OF GROUND BEEF PATTIES

ABSTRACTGround beef patties were formulated with 20% fat, formed on a pattie machine at 113g each (4/lb) and frozen either by liquid nitrogen or liquid carbon dioxide in a cryogenic tunnel at ‐74°C or by a walk‐in mechanical freezer at ‐29°C. Composition, TBA numbers, shrinkage and taste panel scores evaluated at 0, 30, 60, 90 and 120 days after manufacture showed a significant reduction in quality for the mechanically frozen patties when compared with the cryogenic methods. There was no difference between liquid nitrogen and liquid carbon dioxide for quality retention by ground beef patties.

Viability and function of platelets frozen at 2 to 3 C per minute with 4 or 5 per cent DMSO and stored at -80 C for 8 months.

Each of 15 healthy male volunteers was treated with 650 mg of aspirin 24 hours before the autologous transfusion of one unit of freeze-preserved platelets. Freeze-thaw-wash recovery values in vitro, viability and function in vivo, and the bleeding time and platelet aggregation response were measured. The platelets were frozen with 4 or 5 per cent dimethylsulfoxide (DMSO) at an overall rate of 2 to 3 C per minute and were stored at -80 C in a mechanical freezer for up to eight months. They were washed by dilution/centrifugation. The mean recovery in vitro of platelets frozen with 4 per cent DMSO was 76+/-16%; the value was 64+/-16% for platelets frozen with 5% DMSO. The mean in vivo 51Cr recovery of autologous platelets frozen with 4% DMSO was 34+/-6%, and for platelets frozen with 5% DMSO it was 33+/-7%. In both groups the platelet lifespan was normal. There was a significant reduction in bleeding time after the transfusion of a single unit of autologous platelets preserved with either 4 or 5% DMSO, but no improvement in the aspirin-induced platelet aggregation pattern.

4 - Frozen Red Cells

The technical limitations on platelet shelf life and storage have driven research for alternatives, including cryopreservation of platelets. Over the past 60 years, product development has adopted freezing platelets in 6% dimethyl sulfoxide (DMSO) and storage in mechanical freezers at −80 °C for up to 2 years. Frozen platelets show a primed, hypercoagulable in vitro phenotype post-thaw when assayed using morphology, flow cytometry for marker expression, and thrombin capacity. In vivo studies show a role for frozen platelets in the maintenance of hemostasis and data from limited clinical trials show frozen platelets are safe and appear beneficial. As research continues to address the functional role of in vitro assays for clinical outcomes, frozen platelet product development represents a good alternative to room temperature platelets for many applications.

Chapter 15 A Simple Device and Procedure for Successful Freezing of Cells in Liquid Nitrogen Vupor

Cell preservation at low temperatures has become an essential practice in cell biology, particularly in laboratories working with tissue cultures. Sufficient experience has been gained in the practical technology of cryobiology to permit highly successful preservation of cell suspensions. Some of the freezing equipment that has been used for cell freezing includes sophisticated controllers. For the laboratory that only occasionally is required to freeze cells, the use of elaborate and expensive equipment would likely not be justified. Alternative approaches involve the placing of ampoules for timed periods in Dry-Ice chests or Dry-Ice-ethanol mixtures. Other freezing methods involve direct cooling in vapor or liquid nitrogen. In another approach, a mechanical freezer or liquid nitrogen is used to cool an ethanol bath containing the ampoules. While many of the various methods mentioned are reasonably effective, the recovery rate varies largely, according to the rate of initial freezing. The methods also differ in cost, sophistication and automation, amount of handling, and reproducibility of results. The cell freezer to be described has a number of attractive features that appeal to cell biologists and others working with isolated cell suspensions, especially where there is an infrequent need to freeze cells.

CRYO-PRESERVATION OF THE PARASITIC PROTOZOA

In the present paper, about 200 literatures on the cryo-preservation of the parasitic protozoa have been surveyed, and the following problems have been discussed : cooling rate, storage periods at various temperatures, effects of cryo-protective substances in relation to equilibration time or temperatures, and biological properties before and after freezing. This paper is composed of three main chapters, and at first, the history of cryo-preservation is reviewed in details. In the second chapter, other literatures, which were not cited in the first chapter, are introduced under each genera or species of the protozoa. In the last chapter, various factors on cryo-preservation mentioned above are discussed by using the author's data and other papers in which various interesting problems were described. The following conclusions have been obtained in this study : Before preservation at the lowest storage temperature, it appears preferable that samples are pre-cooled slowly at the rate of 1 C per minute until the temperature falls to-25 to-30C. The cooling rate might be obtained by the cooling samples for 60 to 90 minutesat-25 to-30 C freezer. For storage, however, lower temperatures as low as possible are better for prolonged storage of the samples. Many workers recommended preservation of the samples in liquid nitrogen or in its vapor, but the storage in a dry ice cabinet or a mechanical freezer is also adequate, if the samples are used within several weeks or at least several months. Cryo-protective substances such as glycerol or DMSO are highly effective to keep higher survival rate of the protozoa in frozen state. Most workers recommended to use 10% glycerol or 5 to 7.5% DMSO for this purpose. For the use of glycerol, at least 30 to 60 minutes of equilibration at temperatures as high as 37 C, is necessary to produce satisfactory results, because at 25 C or lower temperatures, the cryo-protective action of glycerol becomes insufficient. In DMSO, however, samples should be cooled as soon as possible after adding the substance into protozoan suspension. Prolonged equilibration with DMSO is apparently toxic to trichomonad. Many workers pointed out that biological properties of the protozoa such as infectivity, virulence, antigenicity, and drug resistance, were not changed by prolonged period of preservation at the low temperature. These aspects are greatly advantageous for cryo-preservation. By adopting the cryopreservation technique, furthermore, we save expenses for the maintenance through animal passages or in vitro culture, and thus we can store much more protozoan species or strains in the laboratory. Finally, the author proposed to build the cryo-preservation center of protozoan strains, because in this country we have no adequate center to deposit our strains. Even if nobody use a certain strain for experiment at the moment, yet we can preserve them for the future need. Other laboratories also have many strains, and some of them may be used frequently, but others are not. If all strains be collected in one center, and if the center supplies each strain in case of need, we could save a lot of expense to preserve strains in each laboratory.

Simplified storage and retrieval of stock cultures.

Multiple replicate units of stock cultures of certain bacteria and yeasts can be prepared, stored, and retrieved for use with a minimum of effort. The method involves the coating of sterile glass beads with a mixture of equal parts of broth culture and horse blood, and storage of the coated beads in a single, labeled, plastic tube in a mechanical freezer.

Some variations in lymphocyte freezing methods which do not affect cell viability.

Several variables which differed among lymphocyte freezing and storage protocols, including resuspending media, freezing containers, sealing methods, freezing apparatus, and storage units, were investigated. Loss of viability was variable with the freezing and thawing techniques used; however, in general, lowered viability was not dependent upon the variables tested. Good results were achieved with 3 different, inexpensive freezing devices: the modified‐Hauschka box, a mechanical freezer set at — 68° C, and a Linde BF‐5 biological freezer. Lymphocyte storage in the mechanical freezer proved satisfactory for as long as 5 months.

Simplified Storage and Retrieval of Stock Cultures

Multiple replicate units of stock cultures of certain bacteria and yeasts can be prepared, stored, and retrieved for use with a minimum of effort. The method involves the coating of sterile glass beads with a mixture of equal parts of broth culture and horse blood, and storage of the coated beads in a single, labeled, plastic tube in a mechanical freezer.

Freezing and storing of monkey and rabbit kidney cells for tissue cultures

Summary Cercopithecus monkey kidney cells were more table if frozen from primary cultures than those from fresh trypsinizations when dimethyl sulfoxide in medium 199 was used as the additive. No such difference could be seen with rabbit kidney cells frozen in the same conditions. Rhesus monkey cells were less viable if frozen from primary cultures than those from fresh trypsinizations when glycerol in Eagle's basal medium was used as the additive. Cercopithecus monkey kidney cells showed a half-life of 5 months when stored in a mechanical freezer at −85°C. The longer the period of storing was, the less the number of cultures could be mitated and the later the confluency was reached in culture. The time during which these cells are kept in the fusion zone (0.5 to 40 min) of the freezing curve did not affect the viability of the cells.

Fluorescent Antibody Reactions against Six Species of Simian Malaria in Monkeys from India and Malaysia

Sera from 45 Malaysian and 51 Indian monkeys were tested for fluorescent antibody response to antigens of Plasmodium fieldi, P. gonderi, P. inui, P. coatneyi, P. knowlesi, and P. cynomolgi bastianellii. A total of 17 Malaysian and 33 Indian monkey sera had positive antibody responses with the larger number reacting with P. fieldi and P. gonderi. The results suggest the possibility of prior malaria infection or of nonspecific antibody to other parasites. The intensity of this response to heterologous or common antigens may be sufficient to mask a species-specific response. The use of monkeys having no initial fluorescent antibody titer for Plasmoclium is suggested for studies involving antibody in monkey malaria. The staining of monkey malaria parasites using the fluorescent antibody (FA) technique was first reported by Ingram et al. (1961) using Plasmodium cynomolgi bastianellii. Additional studies by Tobie and Coatney (1961) and Tobie et al. (1962) showed cross-reactions between P. vivax and P. cynomolgi bastianellii. Reported here are the fluorescent antibody reactions of monkey sera from Malaysia and India against six different species of monkey malaria. MATERIALS AND METHODS Thin blood films, containing the parasites of P. fieldi, P. gonderi, P. inzi, P. coatneyi, P. knowlesi, and P. cynomolgi bastianellii, were prepared and stored without fixation at -70 C in a mechanical freezer. Prior to use, they were raised to room temperature in a desiccator and then fixed for 30 min in acetone at -20 C. The slides were dried and then dehemoglobinized by immersion for 5 min in 0.1% HC1, followed by 1-min rinses each in distilled water and phosphate-buffered saline (PBS), pH 7.0. The antigen slides were then ready for immediate use. Rabbit antiserum (globulin fraction) to monkey globulin conjugated with fluorescein isothiocyanate was obtained commercially (Sylvana Chemical Company) and prepared by absorption once with rabbit liver powder and once with rabbit bone marrow powder, and stored at 1: 40 in PBS at -20 C. The 1: 40 dilution was used throughout the studies. Received for publication 28 July 1964. 81 A Leitz SM fluorescence microscope with an Osram HBO-200 light source was used with BG-12 and UG-2 exciter filters and a Wratten 2A barrier filter. For each test, several drops of the serum dilution to be tested were applied to the antigen slide and allowed to react for 20 min at room temperature in a moist chamber. The slide was then given 3 5-min rinses in PBS. Several drops of the labeled rabbit antimonkey globulin were applied and allowed to react for 20 min. The slide was again given three 5-min rinses in PBS and then mounted under a cover slip in buffered glycerine. Fluorescence was graded 1+ to 4+. A reading of 2+ was considered positive and reproducible. Each serum sample was titrated simultaneously against all six antigens. The test sera were from several species of Macaca and Presbytis monkeys collected in the Selangor, Pahang, and Perak states of Malaysia in 1961, and from Macaca mulatta monkeys arriving in our laboratory from New Delhi, India, in 1963. Of the Malaysian monkeys, only one (C-22) was infected with Plasmodium (species undetermined) at the time of collection. Since these were field-collected sera, slides were made only the 1 day. It is therefore impossible to determine if other of these monkeys were suffering from chronic infections. None of the Indian monkeys had malarial infections as determined by the microscopic examination of blood films taken on two separate days. Previous laboratory studies had shown that monkeys having chronic infection with Plasmodium sp. had FA end points of 1: 40 or greater. Even though all the sera were tested starting at the 1: 10 dilution, it was decided to report only those FA reactions of 1 : 40 or greater as being positive. This content downloaded from 157.55.39.249 on Wed, 03 Aug 2016 05:47:52 UTC All use subject to http://about.jstor.org/terms 82 THE JOURNAL OF PARASITOLOGY, VOL. 51, NO. 1, FEBRUARY 1965 TABLE I. Fluorescent antibody test reactions (expressed as reciprocal of titer) of 17 positive sera from Presbytis sp. and Macaca sp. monkeys collected in Malaysia in 1961. Monkey Monkey Species of Plasmodium species no. species no. P. fieldi P. gonderi P. inui P. coatneyi P. obscura 0-61 40 40 P. cristatus C-18 40 C-22 40 40 -25 40 160 C-27 40 C-28 40 40 -29 40 40 C-31 40 C-32 160 C-38 40 M. irus M-312 40 -313 80 M-314 40 M-315 40 M-316 40 M. nemestrina N-93 80 N-94 40 Positive responses 13 4 1 4 Control sera from monkeys known to be free of previous malarial infection gave negative responses. In addition, the labeled antimonkey globulin alone failed to give any response. Monkeys having previous malaria reacted strongly both to homologous and heterologous antigens.

Survival Rates of Rapidly Frozen Bovine Spermatozoa

Summary Subsamples of semen from each of two ejaculates from each of 12 bulls were extended with a yolk-citrate-glycerol extender, equilibrated 5 to 6 hr. at 5° C., and placed directly in a freezing bath at temperatures of +5, 0, −5, −10, −15, −20, −25, −30, −35, and −40° C. The freezing bath was cooled at the rate of 0.8° C. per minute from +5 to −15° C., at a rate of 5° C. per minute from −15 to −40° C., and at the rate of 10° C. per minute from −40 to −75° C. The percentage of motile spermatozoa in ampules subsequently stored in a mechanical freezer at −85° C. for 60 days were, in order of decreasing temperature of the freezing bath at the time of transfer, 29, 21, 24, 27, 33, 36, 35, 35, 35, and 35. From these data it is concluded that time can be saved and a higher percentage of motile spermatozoa recovered by transferring the semen abruptly from +5° C. to a freezing bath ranging from −20 to −40° C.