In vivo distribution of cryogenically preserved guinea pig granulocytes

Granulocytes were isolated from whole blood of guinea pigs by counterflow centrifugation and labeled with [¹⁴C]diisopropylfluorophosphate ([¹⁴C]DFP). One-half of the labeled cells was injected intravenously via the femoral vein into a guinea pig, while the other half was cryogenically preserved with 5% dimethyl sulfoxide (DMSO), 6% hydroxyethyl starch (HES), and 4% human albumin, at a rate of 4 °C per minute by storage at ?80 °C and then stored for 3 days at ?80 °C. Ninety percent of the isolated granulocytes were recovered after cryogenic preservation, thawing, and washing. Aliquots before injection all produced fluorescein from fluorescein diacetate and excluded ethidium bromide. Latex ingestion was 78% and yeast ingestion was 75%. The frozen-thawed-washed-resuspended labeled granulocytes were injected into a second guinea pig. Paired animals sacrificed 35 min after injection were examined in whole-body sections for distribution of radiolabeled granulocytes to the tissues. In two pairs of animals, activity was found in the lung, liver, spleen, and kidney. The technique does not permit a distinction between fresh and cryopreserved granulocytes although there was a greater deposition of fresh cells in the liver and spleen. No activity was found in the blood of the vena cava in animals with either fresh or frozen cells. An animal injected with free [¹⁴C]DFP revealed a vascular distribution with high activity in blood, lung, and kidney, and less activity in the liver and spleen. The data indicate that radiolabeled, cryogenically preserved guinea pig granulocytes exhibited a tissue distribution qualitatively similar to fresh granulocytes, and free [¹⁴C]DFP infused without granulocytes differed qualitatively and quantitatively from fresh and cryopreserved granulocytes.     

Recovery,structure, and function of dog granulocytes after freeze-preservation with dimethylsulfoxide

The recovery, structure and function of dog granulocytes were determined before and after freeze-preservation. Leucocytes were isolated from defibrinated or anti-coagulated whole blood and subsequent erythrocyte sedimentation on a column of 2:1 dextran (6%)-isopaque (33.9%). Granulocytes isolated by these procedures were examined for changes in O₂ consumption associated with phagocytosis, in vitro directed migration (chemotaxis), bactericidal activity, and ultrastructure before and after freezing. Granulocytes were frozen in DMSO (7.5%) and autologous serum or HBSS-minus and 20% autologous serum at the rate of ?1 °C/min to ?80 °C and stored in liquid N₂ vapor.After freeze-preservation, O₂ consumption associated with phagocytosis was decreased by 54 and 64% for granulocytes isolated from defibrinated or from ACD-anticoagulated blood, respectively. Bactericidal activity is only slightly depressed in samples from either isolation method after freeze-preservation when compared to the prefreeze controls, but granulocytes isolated from defibrinated blood are significantly less effective in killing bacteria than those from ACD-anticoagulated blood. Chemotactic response after freeze-preservation was completely inhibited in granulocytes isolated from defibrinated blood. Exposure of granulocytes to ACD inhibited chemotaxis prior to freezing, but the granulocytes responded chemotactically after freeze-thaw and additional washing. The ultrastructure of granulocytes observed before and after freeze-thaw was similar for cells isolated by both methods. However, nuclear, cytoplasmic, and granular changes observed were slightly greater in granulocytes isolated from defibrinated blood. Dog granulocytes isolated by either method withstood freeze-preservation in DMSO to a degree not previously reported.It is concluded that dog granulocytes freeze-preserved by these methods are functional in vitro, but that phagocytic, directed migration, and bactericidal functions and ultrastructure are impaired to different degrees, according to the method of isolation and preparation for storage. These results indicate the need for continued investigation on the effects of storage variables on the preservation of granulocytes.     

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 Ca²⁺-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 × 10⁶ ? 50 × 10⁶) 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.     

Viability of frozen rat granulocytes and granulocyte precursors

Mature rat polymorphonuclear leukocytes (PMNs) were frozen to ?196 °C, thawed, and tested for functional viability using a variety of criteria. The assays for functional viability included: qualitative and quantitative nitroblue tetrazolium tests for phagocytic activity, fluorometric tests for membrane integrity, chemotaxis, and bactericidal activity. Maximal survival was obtained when mature PMNs were frozen in the presence of 10% dimethyl sulfoxide (Me₂SO) and 5% hydroxyethyl-starch (HES) for cells cooled at ~10 °C per minute, followed by rapid warming. Maximal survival was obtained for granulocyte precursor cells (as measured by CFU-c) after freezing in the presence of 10% Me₂SO and cooling at ~10 °C per minute. The principal new findings for mature PMNs were: (i) there was a synergistic effect between intra- and extracellular protective additives; (ii) the optimal cooling rate increases from approximately 0.3 to 10 °C per minute when an extracellular protective agent, such as HES is included in the freezing media; (iii) the zwitterion buffer Hepes has a small but consistently beneficial effect on survival; (iv) granulocytes obtained from peripheral blood consistently show a higher functional survival after freezing (95%) than do PMNs obtained from a glycogen-induced peritoneal exudate (70%); (v) neither serum, plasma, nor other macromolecules are needed in the postt-haw dilution media to obtain high survival; and (vi) cells frozen using an optimized two-step protocol survived as well as those frozen using a continuous cooling protocol.     

Long-term cryopreservation of dog granulocytes

Granulocytes isolated by counterflow centrifugation elutriation (CCE) from leukapheresed dog blood, frozen in liquid nitrogen at ?196 °C, were studied. The effects of long-term cryopreservation on cell recovery and in vitro function were detertmined. In seven separate experiments, an average of 1.7 × 10⁹ granulocytes were obtained. The white cell differential count was 91% granulocytes and 9% mononuclear cells. There was less than 5% red cells presrent and no platelets. Granulocytes were placed in Hemoflex bags and mixed slowly with equal volumes of sterile ice-cold hyperosmolar cryoprotectant buffer to make a final composition of 5% dimethylsulfoxide (DMS), 6% hydroxyethyl starch (HES), and 4% bovine serum albumin (BSA), pH 7.1. Total volumes of 40 ml were frozen at a cooling rate of 4 °C per minute and stored for periods of 1, 34, 60, 90, and 132 weeks in liquid nitrogen at ?196 °C. Thawing was done at a rate of 190 ° per minute to 10 °C. The recovery of cells was 95%, 105%, 100%, 100%, and 88% respectively. Ethidium bromide exclusion, indicative of viable nuclei, was 91%, 81%, 94%, 89%, and 80% respectively. Virtually all thawed cells ingested opsonized Fluolite particles, but the number ingested was approximately one-half that of prefreeze values. Thawed cells also demonstrated superoxide anion synthesis at rates approximating those in unfrozen granulocytes. These results indicate that dog granulocytes obtained by leukapheresis may be preserved in liquid nitrogen at ?196 °C with high cellular recovery and at least 50% phagocytic function.     

Liquid and cryopreservation effects on in vitro function of dog granulocyte concentrates prepared for transfusion

The effects of liquid and Cryopreservation on in vitro function of dog granulocyte concentrates prepared by continuous flow centrifugation leukapheresis and counterflow centrifugation elutriation are presented. These homogeneous granulocyte concentrates (97 ± 2% granulocytes, 99.4 ± 0.3% viable) were cryopreserved in 5% DMSO and 5% HES dissolved in 2 g% BSA, 20% autologous citrated plasma in a modified a minimal essential medium. The granulocyte recovery was 87.6 ± 2.4% relative to the number of intact and viable granulocytes in the washed suspension of cells. In vitro functions of chemotaxis, phagocytosis, bactericidal activity, and selected enzymes were not affected by 12–24 hr storage at 4–6 °C. Frozen, thawed, and washed granulocytes showed a significant decrease (P < 0.01) in chemotactic recognition and response but not chemokinetic response, although it was depressed. Phagocytosis of latex beads and associated burst of O₂ consumption also decreased significantly (P < 0.05) to approximately 50% of the original prefreezing value. However, the killing of live Escherichia coli was not depressed to the extent expected and suggested by loss of O₂ consumption and selected enzyme activity. The cryopreservation of viable homogeneous granulocyte concentrates in sufficient quantity for transfusion in the neutropenic and/or septicemic dog model is demonstrated in these results.     

Freeze preservation of platelets using hydroxyethyl starch (HES); a preliminary report.

A comparative study has been made of platelets stored by freeze preservation following treatment with dimethyl sulfoxide (DMSO) or hydroxyethyl starch (HES) with fresh platelets and platelets stored at 4 °C for 48 hr. The indices studied were platelet recovery, pH, light microscope morphology, platelet Factor 3 (PF₃) availability and the hypotonic stress response. The DMSO preserved platelets gave a better response to hypotonic stress and incurred lesser degrees of membrane damage as demonstrated by PF₃ availability. There was however a significantly higher recovery of platelets treated with HES; with DMSO the osmotic damage inflicted during removal caused considerable lysis. Platelets frozen by DMSO or HES gave consistently better in vitro results than platelets stored at 4 °C for 48 hr. A preliminary clinical trial of HES preserved platelets has confirmed haemostatic effectiveness in vivo. HES being relatively nontoxic, platelets can be infused immediately after thawing and with minimal post thaw manipulation, thus maintaining a relatively closed system. It is concluded that cryopreservation with HES is a practical and effective means for long term platelet storage.     

Morphology of glutaraldehyde-fixed preserved red blood cells and 24-hr post-transfusion survival

Liquid-stored red blood cells and washed, previously frozen red blood cells were studied to determine whether a correlation existed between morphology and post-transfusion survival. Red cell concentrates were stored at 4 °C in citrate-phosphate-dextrose (CPD) for 21 days or in CPD-adenine (CPDA-1, CPDA-2, or CPDA-3) for as long as 35 days as liquid-preserved red cells. Both nonrejuvenated and rejuvenated red blood cells were frozen with 40%$\text{w}\text{v}$ glycerol at ?80 °C and were washed prior to testing.Samples of fresh, liquid-stored, and washed, previously frozen red blood cells were fixed with a 2% veronal glutaraldehyde solution. Phase, light, and electron microscopy were used to measure the numbers of discocytes, discoechinocytes, echinocytes, echinospherocytes, and spherocytes in each sample. A morphology score was assigned, with 100 representing all discocytes and 500 all spherocytes. In all samples phase and light microscopy gave nearly identical scores (r = 0.94), and phase and electron microscopy gave highly similar scores (r = 0.83).The morphology score proved to be a good indicator of 24-hr post-transfusion survival in liquid-stored red blood cells but not in washed, previously frozen red blood cells. Red blood cells stored in the liquid state at 4 °C in CPD, CPDA-1, CPDA-2, or CPDA-3 showed a significant inverse correlation between morphology and 24-hr post-transfusion survival (r = ?0.611) and a significant correlation between red cell ATP and 24-hr post-transfusion survival (r = 0.742). We saw no significant correlation between morphology scores and 24-hr post-transfusion values or between ATP levels and post-transfusion survival values in nonrejuvenated or rejuvenated washed, previously frozen red blood cells.     

An experimental study on the freezing of red blood cells with and without hydroxyethyl starch

Red blood cells were frozen in small capillaries down to ?196 °C at different linear cooling rates with or without the cryoadditive HES; the thawing rate was 3000 or 6500 °C/min. Hematocrit and hydroxyethyl starch concentration varied independently. The hemolysis of red blood cells was determined photometrically after 250-fold dilution and compared to totally hemolyzed samples. The typical U-shaped curves for hemolysis as a function of the cooling rate were obtained for all cell suspensions investigated. Relative optimum cooling rates were determined for the respective combinations of HES and hct. The results show that increasing hct causes an increased hemolysis; increased HES concentration C_HES reduces the optimum cooling rate B_opt; increased hct results in higher optimal cooling rates. The findings allow one to establish a linear correlation of the HES concentration and the optimum cooling rates when the dilution of the extracellular medium by the cell water efflux during freezing is taken into account. A comparison with results from larger volumes frozen (25 ml) shows that the established relationship between hematocrit, HES concentration, and optimal cooling rate remains valid.     

Neutrophil migration in canines: In vivo comparison of granulocyte function following 24-hour storage at 6 or 20 °C of leukocyte concentrates or of granulocytes purified by counterflow centrifugation-elutriation

The use of granulocyte-rich concentrates from leukapheresis purified by counterflow centrifugation—elutriation to obtain pure granulocytes for transfusion studies in cyclo-phosphamide-induced neutropenic animal models is reported. Our data for granulocyterich leukapheresis concentrates indicate that room temperature (20 °C) appears to be preferred to 6 °C for short-term granulocyte storage. The data also indicate that although the granulocytes isolated by counterflow centrifugation—elutration may retain in vitro functions of chemotaxis, phagocytosis, and bactericidal activity, the in vivo function of migration into skin chambers for isolated granulocytes is seriously impaired after storage for 18 to 24 hr at both 6 and 20 °C. This loss of in vivo function of stored granulocytes occurs in isolated granulocytes obtained by both counterflow centrifugation-elutriation and dextran sedimentation, and it is not observed in the leukocyte concentrates held at 20 °C. The results of these studies are fourfold. First, freshly isolated granulocytes display no apparent loss of either in vivo or in vitro function. Second, granulocytes isolated by counterflow centrifugation-elutriation or dextran sedimentation and stored at 6 or 20 °C are severely impaired in terms of their in vivo chemotactic function but display no loss of in vitro efficacy. Third, 20 °C storage of granulocyte-rich leukapheresis concentrates for 18 to 24 hr is superior to 6 °C storage. Fourth, in vitro analysis may be limited in its ability to indicate in vivo function as a measure of success in granulocyte preservation studies.     

Comparisons of glucose,sucrose, and dimethyl sulfoxide as cryoprotective agents for Babesia rodhaini, with estimates of survival rates

Comparisons were made between glucose, sucrose, and dimethyl sulfoxide (DMSO) as cryoprotective agents for the hemoprotozoan parasite, Babesia rodhaini, using infectivity for mice as the criterion of survival. Concentrations of the cryoprotectants tested were from 0.1 to 0.5 M for the sugars, and 1.5 to 2.5 M for DMSO. Glucose and sucrose were comparable as cryoprotectants, although glucose reduced infectivity of the parasites slightly more than did sucrose at above-freezing temperatures. When sucrose and DMSO were compared for cryoprotection during cooling to ?196 °C at nominal rates of 5, 100, and 500 °C/min, parasite survival varied with the type and concentration of cryoprotectant, but was higher in blood containing DMSO at all three cooling rates. The percentages of parasites that survived cooling at 100 °C/min and frozen storage in the presence of DMSO ranged from 20 to 36%.     

A simplified method for freezing mouse embryos

Mouse oviducts containing eight-cell embryos were frozen to ?196 °C in 1.45 m DMSO. The cooling rate was 0.3 °C/min and thawing occurred at 3 °C/min. Dilution of DMSO took place either before or after flushing of the thawed oviducts. The yield of intact embryos was higher in the second group.In one particular series involving 21 donor mice (natural ovulation) 88 recovered embryos were transferred to the oviducts of recently mated pseudopregnant mice without prior in vitro culture to the blastocyst stage. Fifty-five live young were born.It is concluded that the freezing of embryos in the oviduct is a reliable method for establishing an embryo bank. Handling and collection of isolated embryos is not required and a large amount of material can be frozen at once. In vitro culturing of embryos is not required immediately after thawing in order to obtain a high yield of live young.     

Recovery of a marine dinoflagellate following controlled and uncontrolled freezing

A free-living, marine dinoflagellate, Crypthecodinium cohnii, was successfully preserved by controlled and uncontrolled freezing. Tolerance testing to various concentrations of dimethylsulfoxide (DMSO) and glycerol established that 7.5% glycerol was the best cryoprotectant. Controlled freezing was accomplished by using a biological freezer to obtain a 1 °C/min cooling rate. After storage for a minimum of 7 days at ?150 °C material frozen by this method demonstrated a 47.7% mean recovery, and cells were viable through five subcultures. Uncontrolled freezing resulted from placing the ampoules on the bottom of a low temperature refrigerator at ?55 °C for 1 hr. This material demonstrated a mean recovery of 30.8% with a much wider range. Cells were initially nonmotile following recovery, and in those recovered after uncontrolled freezing motility was further delayed. One strain was viable after 6 years of storage with a 68% recovery following controlled freezing.The lack of motility immediately following recovery leads to inaccuracies when determining the percentage of cells recovered. Dilution techniques have been used for nonmotile recovered cells, but this method has been unsuccessful in our laboratory. Delayed motility has been reported for other flagellates and work in our laboratory indicates that flagellar shearing may be the cause.     

Plasmodium falciparum and P. knowlesi: low temperature preservation using dimethylsulfoxide

Erythrocytic stages of Plasmodium knowlesi and P. falciparum have been successfully recovered from the frozen state (i.e., ?196 C) using dimethylsulfoxide (DMSO: 7.5% final concentration, v/v) as the cryoprotective agent. Quantitated preparations of P. knowlesi, stored for 202 days, yielded patent and synchronous infections as early as 72 hr postinoculation. Similar results were obtained with blood samples with P. falciparum.     

Effect of DMSO on the dielectric properties of canine kidney tissue

The dielectric permittivity and conductivity of canine kidney tissue samples were measured at R_f frequencies between ?20 °C and +20 °C. Some of the kidneys had been perfused with DMSO (10%) in canine plasma, others with physiological saline alone. The DMSO greatly increases the conductivity of frozen tissue above that of tissue not treated with this cryoprotectant. Apparently, the chief reason for nonuniform heating of a partially frozen organ in a microwave field is the great change in tissue conductivity as it thaws. We suggest that the effect on the conductivity of tissue should be considered in the choice of a cryoprotectant for tissues which are to be thawed by microwave or radiofrequency irradiation.     

Interaction of rate of latent heat removal during freezing and rates of subsequent cooling and warming on survival of the blood protozoan, Babesia rodhaini

Babesia rodhaini parasites in murine blood containing 1.5 m DMSO were frozen at two rates, as judged by the duration of the “freezing plateau”, then cooled to ?196 °C and rewarmed at two rates to detect interactions between the duration of the plateau and rates of subsequent cooling and rewarming. Infectivity tests showed that fast and slow freezing (plateau times of about 1 sec and 30 sec, respectively) had similar effects on parasite survival when cooling was at 130 °C/min and warming was at 800 °C/min. However, when either the cooling rate was increased to 3500 °C/min or the warming rate was decreased to 2.3 °C/min, fast freezing decreased parasite survival more than did slow freezing. It is suggested that fast freezing accentuated the damaging effects of fast cooling and slow warming by increasing intracellular ice formation.     

Cell separation in the buffy coat

One of the most rapid methods to determine cell counts in whole blood is by way of layer thickness measurements of the buffy coat. The purpose of this study was to determine the separation and purity of blood cells in the different layers of the buffy coat. Blood samples were centrifuged at 10,000 g in microhematocrit tubes with an inserted float to expand the buffy coat region. Whole blood from normal laboratory individuals separates by density into four regions: platelets, a layer of lymphocyte and monocytes, granulocytes and erythrocytes. A thin band of highly swollen red cells was discovered between the buffy coat layers of many normal volunteers and patients. Stereological analysis of electron micrographs showed that mixing of formed elements within the layers is less than 2% with the exception of some erythrocytes, which can make up a higher volume fraction in the lymphocyte/monocyte and granulocyte layers. The red cell column contains about 95.7% erythrocytes and is depleted of platelets and leukocytes. In approximately 5% of hospital blood samples, the granulocyte-erythrocyte interface was feathered and undetectable, and a significantly higher volume fraction of red cells was found among the granulocytes. Cell mass density determinations indicate that the erythrocytes in these abnormal granulocyte layers have a lowered mass density, overlapping with that of the granulocytes.     

E-rosette dormation of fresh versus frozen-thawed lymphocytes from leukemic patients and normal individuals.

Peripheral blood lymphocytes from normal individuals and patients with ALL, AML, CLL, CML and Hodgkin's disease were compared in the fresh and frozen-thawed states. The cells were frozen in DMSO by either a controlled (1 °C/min) or uncontrolled rate freezing process and stored in liquid nitrogen (?196 °C) for 1 day to 12.5 years. No significant difference was found between fresh and frozen-thawed cells of normal or leukemic subjects in the observed percentage of spontaneous E-rosette formation. Leukemic cells frozen by the controlled rate method and preserved in liquid nitrogen for 12.5 years were found to be fully capable of rosette formation, and when stained with Trypan Blue, the majority (> 70%) were found to be viable.     

Non-toxic freezing media to retain the stem cell reserves in adipose tissues

Subcutaneous adipose tissue is a rich source of stromal vascular fraction (SVF) and adipose-derived stromal/stem cells (ASCs) that are inherently multipotent and exhibit regenerative properties. In current practice, lipoaspirate specimens harvested from liposuction surgeries are routinely discarded as a biohazard waste due to a lack of simple, cost effective, and validated cryopreservation protocols. The aim of this study is to develop a xenoprotein-free cryoprotective agent cocktail that will allow for short-term (up to 6 months) preservation of lipoaspirate tissues suitable for fat grafting and/or stromal/stem cell isolation when stored at achievable temperatures (−20 °C or −80 °C). Lipoaspirates donated by three consenting healthy donors undergoing elective cosmetic liposuction surgeries were suspended in five freezing media (FM1: 10% DMSO and 35% BSA; FM2: 2% DMSO and 43% BSA; FM3: 10% DMSO and 35% lipoaspirate saline; FM4: 2% DMSO and 6% HSA; and FM5: 40% lipoaspirate saline and 10% PVP) all suspended in 1X DMEM/F12 and frozen using commercially available freezers (−20 °C or −80 °C) and stored at least for a 1 month. After 1 month of freezing storage, SVF cells and ASCs were isolated from the frozen-thawed lipoaspirates by digestion with collagenase type I. Cell viability was evaluated by fluorescence microscopy after staining with acridine orange and ethidium bromide. The SVF isolated from lipoaspirates frozen at −80 °C retained comparable cell viability with the tested freezing media (FM2, FM3, FM4) comparable with the conventional DMSO and animal serum media (FM1), whereas the FM5 media resulted in lower viability. In contrast, tissues frozen and stored at −20 °C did not yield live SVF cells after thawing and collagenase digestion. The surface marker expression (CD90, CD29, CD34, CD146, CD31, and CD45) of ASCs from frozen lipoaspirates at −80 °C in different cryoprotectant media were also evaluated and no significant differences were found between the groups. The adipogenic and osteogenic differentiation potential were studied by histochemical staining and gene expression by qRT-PCR. Oil Red O staining for adipogenesis revealed that the CPA media FM1, FM4 and FM5 displayed robust differentiation. Alizarin Red S staining for osteogenesis revealed that FM1 and FM4 media displayed superior differentiation in comparison to other tested media. Measurement of adipogenic and osteogenic gene expression by qRT-PCR provided similar outcomes and indicated that FM4 CPA media comparable with FM1 for adipogenesis and osteogenesis.     

The Migration Test on Circulating Bovine Leukocytes and its Possible Application in the Diagnosis of Johne's Disease

The leukocyte migration test for in vitro studies of delayed type hypersensitivity has recently been reviewed (Søborg & Ben-dixen 1967; Bendixen & Søborg 1969). Søborg & Bendixen applied the test to circulating leukocytes in man and thereby widely increased the potentialities of this test. They obtained high leukocyte yields with only moderate erythrocyte admixture by harvesting the supernatant plasma after sedimentation of the erythrocytes for 60 min. at 37°C in the normal gravitational field (1 × g)- Their procedure was unsuitable for the present investigation because bovine erythrocytes sediment so slowly. Sedimentation after clumping at the interphase of aqueous solutions of polymers, dextran and methylcellulose, in combination with metrizoic acid (Böyum 1968) was tried without success because the vast majority of the leukocytes sedimented together with the erythrocytes. Separation of leukocytes from erythrocytes could not be achieved by differential centrifugation.