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.     

Changes in the lipid composition of embryonic cells of the mussel Mytilus trossulus during cryopreservation

A comparative analysis of the lipid composition of embryonic cells of the mussel Mytilus trossulus prior to, and after, cryopreservation in liquid nitrogen was carried out in the presence of two types of cryoprotectors: 1) dimethylsulfoxide and trehalose; and 2) dimethylsulfoxide, trehalose, total lipid extract from the mussel Crenomytilus grayanus, alpha-tocopherol and ascorbic acid. It was found that not only the cell viability but also the fatty acid composition of cell lipids after cryopreservation depend on the composition of cryoprotectors used. The content of saturated fatty acids, monoenic and polyenic fatty acids, the omega 3/omega 6 ratio, and the index of nonsaturation in the fatty acid composition of lipids was shown to change remarkably after cryopreservation. Possible reasons of these changes are discussed.     

Unfractionated human marrow cell cryopreservation using dimethylsulfoxide and hydroxyethyl starch

Unfractionated bone marrow (BM) cells were cryopreserved in 1- to 2-ml aliquots using a mixture containing both 5% dimethylsulfoxide (DMSO) and 6% hydroxyethyl starch (HES) in an attempt to increase the viable cell yield and reduce the clumping after thawing, observed when 10% DMSO is used alone. Samples thawed after storage for 6 months in the vapor phase of liquid nitrogen, were assayed. Compared to prefreeze values, there was both a greater number of cells that excluded Trypan Blue (50 +/- 12 vs 28 +/- 12%, P less than .01) and a greater CFU-C Recovery (110 +/- 20 vs 89 +/- 35%, P less than .02) for cells in the DMSO/HES mixture, compared to those in 10% DMSO alone. No macroscopic clumping of the thawed cells was observed for those cryopreserved in the mixture in contrast to those in DMSO alone. Freezing was done without a rate-controlled freezing apparatus by simply placing the samples initially into a -80 degrees C freezer, and then later into a liquid nitrogen freezer. Additional samples stored in the DMSO/HES mixture were kept at only -80 degrees C, and when thawed 12 to 16 months later also gave an excellent CFU-C recovery (105 +/- 39% of prefreeze). The DMSO/HES mixture allows for a simplified BM cryopreservation technique that not only assures excellent recovery of CFU-Cs and eliminates clumping upon thawing, but also does not require either the use of a rate-controlled freezer or liquid nitrogen temperatures for storage up to a year.     

Factors in the cryopreservation of bone marrow cells from children with acute lymphocytic leukemia.

Bone marrow cells from 42 children with acute lymphocytic leukemia in remission and 19 normal adults were preserved in liquid nitrogen for periods of up to 50 weeks. Many factors in the process of cryopreservation were investigated in an attempt to optimize the recovery of the bone marrow colony forming cells. The effect of cryopreservation on the cells which produce colony stimulating activity also was investigated. With optimization of this technique, it was observed that approximately 100% of the bone marrow nucleated cells were recovered and approximately 50% of the total colony forming cells were viable after 50 weeks in liquid nitrogen.     

On the mechanism of cryoprotection by polyethylene oxide.

Bone marrow cells and peripheral blood leukocytes have been exposed to a 15% solution of polyethylene oxide of molecular weight 400 (PEO-400) and the effect on DNA synthesis, oxygen uptake, and the activity of oxidative-reductive enzymes has been studied. All the metabolic activities studied were reduced during exposure to PEO-400, but in each case the effect was completely reversed when the cryoprotective agent was removed. It is suggested that the cryoprotective action of PEO-400 may be linked with its ability not only to dehydrate cells but also to depress oxidative metabolism.     

Changes of soluble sugars and ATP content during DMSO droplet freezing and PVS3 droplet vitrification of potato shoot tips

The potato's great genetic diversity needs to be maintained for future agricultural applications and can be preserved at ultra-low temperatures. To decipher detailed physiological processes, the aim of the study was to analyze the regrowth in 28 gene bank accessions and to reveal metabolite changes in a subset of four accessions that showed pronounced differences after shoot tip cryopreservation using DMSO droplet freezing and PVS3 droplet vitrification. Regrowth varied in all 28 genotypes ranging from 5% (‘Kagiri’) to 100% (‘Karakter’) and was higher after PVS3 droplet vitrification (71 ± 19%) than after cryopreservation using DMSO (54 ± 17%). Sucrose, glucose, and fructose were analyzed and showed significant increases after pre-culture in combination with PVS3 or DMSO and liquid nitrogen treatment and were reduced during regeneration. In contrast, adenosine triphosphate (ATP) reached its minimum concentration after cryoprotection and liquid nitrogen treatment and recovered most quickly after PVS3 droplet vitrification. A shortening of the explant pre-culture period reduced dramatically the regrowth after PVS3 vitrification. However, correlations between the shoot tip regrowth and sugar concentration were absent and significant at a low extent with ATP (r = 0.4, P < 0.01). Interestingly, several sub-cultivations of the donor plants from the previous stock affected negatively the regrowth. In conclusion, the cryopreservation protocol, genotypes, pre-culture period and number of sub-cultures affect the regrowth ability of explants, which was best estimated by the ATP concentration after low-temperature treatment. Due to the superior performance of PVS3, the routine potato cryopreservation at the Gatersleben gene bank was changed to PVS3 droplet vitrification.     

Long-term preservation of canine bone marrow: in vitro studies.

In vitro studies were performed on canine bone marrow frozen with DMSO and stored in liquid nitrogen for 2 to 6 months. The results are compared with previously reported parallel in vivo experiments that demonstrated no loss of stem cells. When studies were performed immediately after thawing, there was no substantial drop in the count of nucleated cells and, except for megakaryocytes, there was no alteration of the bone marrow morphology. After two washes, and removal of DMSO, the nucleated cell count dropped to 50% of its previous value. Optic and electron microscopy showed severe damage in mature myeloid elements. In some instances, the cells had a condensed nucleus similar to the red-purple inclusion body of LE cells (as observed in systemic lupus erythematosus), and electron microscopy showed heavy chromatin clumping. On the other hand, both optic and electron microscopy showed a good preservation of lymphocytes, plasmocytes, and erythroid precursors. Two-hour DNA synthesis slightly dropped after storage, and this drop appeared more consistent when related to a constant volume of bone marrow (50 microliters) rather than to a constant number of nucleated cells (10(6)). In five instances frozen and thawed bone marrow was grown in short-term cultures, and analysis of 98 metaphases showed no major aberrations of the chromosomes and only 2% of minor aberrations, such as breakages and fragments. These data, compared with the results of previous in vivo experiments that showed no loss of stem cells after 5 months storage, suggest that stem cells are less sensitive to freezing and thawing injury than myeloid elements and/or that it might be safer for the thawed bone marrow not to be manipulated before infusion.     

Ultrastructure of the lymphocytes in lymphoid organs following cryopreservation with PEO-400 protection

Electron microscopic examination of lymphocytes after cryopreservation with PEO-400 used as a cryoprotectant has shown that changes in the cytoplasmic membrane structures are of reversible character after thawing. Programmed freezing with a period of temperature holding is beneficial for lymphocyte cryopreservation.     

Recovery of CFU-GM after freezing of normal human bone marrow: effect of three dilution techniques after thawing

In the cryopreservation procedures intended for autotransplantation of human bone marrow a controversial point is represented by the methods of reconstruction of the cellular suspension after thawing and before infusion into the patient. To evaluate how the dilution rate after thawing affects bone marrow viability, we cryopreserved the bone marrow from 16 hematologically normal patients in DMSO at a concentration of 10%. After thawing, the cells were diluted according to three different techniques and their viability was tested by the growth of CFU-GM in methylcellulose. The average recovery of CFU-GM, in comparison with that of fresh cells, was satisfactory and not affected by the type of dilution. In conclusion, if we accept that the resistance to osmotic stress due to the cryoprotectant is similar for stem cells and CFU-GM, we can maintain that a slow, gradual dilution is not a necessary condition to assume the staminality of bone marrow designed for autotransplantation.     

The effect of cryopreservation on the cytogenetic characteristics of a cell subline of skin fibroblasts from the Indian muntjac

After thawing cells, previously cryopreserved in the presence of dimethyl sulfoxide (DMSO), a decrease in their viability and increase in unscheduled DNA synthesis was observed. In 7 days, these parameters restored to the control level. Cryopreservation without DMSO resulted in the decrease in both cell viability and replicative and unscheduled DNA synthesis. In 14 days, these characteristics were seen to return to the normal level. Cryopreservation of cells without DMSO and their preservation in liquid nitrogen induced the frequency of chromosomal aberrations, mostly chromosomal breaks. The frequency of chromosomal aberrations increased with the duration of cell preservation in liquid nitrogen. The normal level was achieved following 7 days after cell thawing. Cells treated with DMSO only (without cryopreservation) display an increased number of chromosomal and chromatid breaks and translocations. Nonrandom distribution of chromosomal aberrations was observed, with particular chromosomes being involved in the appearance of dicentrics and translocations. The data obtained indicate that cryoprotective activity of DMSO is probably associated with the cell repair systems. The detected antimutagenic and mutagenic activity of DMSO may presumably reflect various conditions for its interaction with cells (with or without cryopreservation), as well as it may be specific for the muntjac cell line used in the present work.     

The mechanism of action of low temperature and cryoprotective agents on immunoproteins

The freezing and thawing of specific antitoxic serum proteins without a cryoprotective agent results in a reduction of titer. A freezing regimen has been chosen and the cryoprotectant PEO-400 has been added to prevent a reduction in activity of antitetanus serum after 6 months of storage in liquid nitrogen. The study of dielectric properties of proteins in solution and measurements of spin-label rotational diffusion have made it possible to estimate the degree of dehydration of protein molecules in the presence of PE-400. We suggest that a damping coat of PEO forms around protein molecules at the expense of hydrophilic and hydrophobic interactions. We further suggest that this damping coat is one of the main mechanisms of cryoprotective action of polyethylene oxide, and possibly of other cryoprotectants such as glycerol.     

Collection,processing and freezing of equine bone marrow cells

There is no consensus on aspects of equine bone marrow collection and processing. The study aimed to describe the collection of large volumes of bone marrow from horses of advanced age, with emphasis on bone marrow mononuclear cells (BMMCs) recovery and viability after cryopreservation. Fourteen horses, aged 3–24 years, were divided into three experiments. E1 studied the feasibility of collecting 200 mL from the sternums of horses of advanced age; E2 examined the number of cells obtained from the first and last syringe of each puncture; and E3 investigated the influence of heparin concentration on the prevention of cell aggregation, and cell viability after freezing in liquid nitrogen. Bone marrow aspirations were done with syringes pre-filled with Iscove's modified Dulbecco's medium and different concentrations of sodium heparin. BMMCs were counted, cell viability was determined, and samples were frozen. Bone marrow collection from the sternum is safe, even at large volumes and from horses of advanced age, and the number of cells recovered decreases with successive aspirations (p < 0.0001). Heparin concentration influenced cell aggregation, and recovered cells continued to be commercially viable after 150 days in frozen storage.     

Leukapheresis cell concentration adjustment required for a successful recovery of HSC after cryopreservation

The recovering of an adequate number of hematopoietic stem cells after cryopreservation is considered pivotal for successful transplantation. Various factors could influence the recovery of HSC following processing and cryopreservation. Therefore, leukapheresis product from thirty patients was cryopreserved in 10% DMSO in cryopreservation bags for their autologous bone marrow transplantation, and 2 ml were cryopreserved in cryovials for post-thaw viability assessment by flow cytometry. The percentage of viable HSCs recovered post-cryopreservation in leukapheresis product was significantly influenced by the concentration of the total nucleated cells cryopreserved per volume. Patients receiving a higher rate of viable HSCs resulted in earlier engraftment of both neutrophils and platelets, so they have been discharged earlier from the hospital. Furthermore, Storage temperature and duration played a role in the recovery of these cells and for the support of the findings, age of the patient at the time of collection did not show any impact on the recovery of this HSC post-cryopreservation. In conclusion, various influencing factors must be taken into consideration during the cryopreservation of HSCs, especially for poor mobilizing patients with a low number of collected hematopoietic stem cells.     

造血细胞活力冷冻损伤的可恢复性

人骨髓冻存后其造血祖细胞活力有一定程度下降,本研究对这种下降的可逆性作了初步观察。结果发现,用双层法和单层法作CFU-GM培养时,未冻存骨髓集落产率相近,冻存骨髓双层法的CFU-GM产率高于单层法。骨髓细胞用20％FM-CM、PHA-LYCM、PHA-PMCM预孵育2h后,分别测定其CFU-GM、BFU-E与CFU-Mix,发现这种孵育过程对未冻存骨髓的集落产率无明显影响,而冻存骨髓的集落产率在孵育后可升高(GEMmeg除外)。说明骨髓造血祖细胞对冻存的损伤反应不均一,部分受损细胞在一定条件下可以恢复其增殖活力。这对于用冻存骨髓作骨髓移植可能有一定意义。     

Cryopreservation of wheat suspension culture and regenerable callus

Wheat (Triticum aestivum L. cv. Norstar) suspension cultures and regenerable calli initiated from immature embryos can be cryopreserved in liquid nitrogen temperature (–196°C) by slow freezing (0.5°C/min) in the presence of a mixture of DMSO and sucrose or sorbitol. Cold hardening or ABA treatment before cryopreservation increased the freezing resistance and improved the survival of wheat suspension culture in liquid nitrogen. Callus culture, established from immature embryos, prefrozen in 5% DMSO and 0.5M sorbitol survived liquid nitrogen storage and resumed plant regeneration after thawing. The results confirm the feasibility of long term preservation of wheat embryo callus by cryopreservation and retention of plant regeneration ability.Abbreviations ABA Abscisic acid - 2,4-D 2,4-Dichlorophenoxyacetic acid - DMSO Dimethylsulfoxide - LN Liquid nitrogen - TTC 2,3,5-triphenyltetrazolium chloride NRCC No. 23850.     

Pigment recovery from encapsulated-dehydrated Vaccinium pahalae (ohelo) cryopreserved cells

Pigment producing in vitro cells of Vaccinium pahalae (ohelo) were tested for their ability to survive cryopreservation and retain pigment-production capacity after encapsulation-dehydration. Preculture of cells for 6 to 8 days in a medium containing 1.0 M sucrose was essential before dehydration. Reduction of bead water content before quenching in liquid nitrogen was highly correlated (r = 0.94) with increased survival rate in cells after cryopreservation. Dehydration of beads for 4 h was satisfactory for survival of cells. After quenching in liquid nitrogen, colored cells became pale, but pigment content was recovered once cells resumed growth. After three subcultures, cells regained their maximum capacity for pigment accumulation. The percentage of colored-to-total cell volume was not influenced by cryopreservation. Encapsulation-dehydration and cryopreservation did not diminish the capacity of cells to produce anthocyanins and other flavonoids. This revised version was published online in June 2006 with corrections to the Cover Date.     

Optimal conditions for cryopreservation of primary chicken embryo kidney cells with dimethyl sulfoxide

Conditions were evaluated for optimum cryopreservation of primary chicken embryo kidney (CEK) cells. The recovery of viable CEK cells was best (50.8% viability) when the concentration of dimethyl sulfoxide (DMSO) in the freezing medium was 20% (v/v). The viability of primary CEK cells was not influenced by the concentration of calf serum in the freezing medium, the duration of storage at −70°C before storage in liquid nitrogen, cell concentration, or the method of addition or dilution of DMSO. Thawed cells recovered and grew in complete growth medium similarly to cells freshly isolated from kidney, and influenza viruses produced plaques in the monolayer. The cryopreservation procedures described here may facilitate maintenance of a standard stock of primary CEK cells for laboratories where preparation of primary CEK cells is not an option.     

Cryopreservation of basidiomycete strains using perlite.

A new alternative method using perlite as a particulate solid carrier in the growth medium with a cryoprotectant was successfully tested for cryopreservation of several basidiomycete species from different genera (Armillaria, Pleurotus, Pluteus, Polyporus) which failed to survive or retain their properties in cryopreservation procedures routinely used in our laboratory. Frozen basidiomycete strains were kept in cryovials submerged in liquid nitrogen and were either immediately after the freezing process or after a 6-month storage thawed and checked for viability, purity and changes in growth, morphology and biochemical characteristics. All cultures survived the cryopreservation procedure and no negative effects of cryopreservation by this method have been observed after 6 months of storage in liquid nitrogen.     

The viability of cryopreserved PBPC depends on the DMSO concentration and the concentration of nucleated cells in the graft

BACKGROUND: DMSO is widely used as a cryoprotectant for PBPC. It is desirable to reduce the amount of DMSO without jeopardizing the quality of the stem cell product. The present study was undertaken to investigate whether recovery and survival of CD34+ cells would be significantly altered when PBPC used for autologous transplantations were cryopreserved with four different DMSO concentrations. METHODS: Apheresis samples of PBPC from 20 consecutive patients were mixed in parallel with 2%, 4%, 5% and 10% DMSO, frozen with identical cell concentrations at a controlled rate, and stored in liquid nitrogen for 6-8 weeks. PBPC samples from 11 consecutive patients were also cryopreserved with two different cell concentrations (150 and 300 x 10(6) nucleated cells/mL) to investigate the effect of increasing the cell concentrations while decreasing the DMSO concentration. The flow cytometric absolute count method, based on ISHAGE guidelines, was used to measure the absolute count of total and viable CD34+ cells in the post-thaw samples. RESULTS: PBPC cryopreserved at 150 x 10(6) cells/mL with 2% DMSO yielded significantly inferior CD34+ cell recovery (P < 0.001) and survival (P < 0.001) compared with cryopreservation with 4% and 5% DMSO. This was also observed when comparing higher cell concentrations. However, a reduced cell survival (P = 0.02) was observed when the nucleated cell concentration was increased from 150 to 300 x 10(6) cells/mL in samples cryopreserved with 5% DMSO. DISCUSSION: We conclude that 5% DMSO may be the optimal dose for cryopreserving PBPC as long as the cells have not been concentrated at much more than 200 x 10(6) nucleated cells/mL.     

Cryopreservation of nucellar cells of navel orange (Citrus sinensis Osb. var. brasiliensis Tanaka) by vitrification

The nucellar cells of navel orange(Citrus sinensis Osb. var. brasiliensis Tanaka) were successfully cryopreserved by vitrification. In this method, cells were sufficiently dehydrated with highly concentrated cryoprotective solution(PVS2) prior to direct plunge in liquid nitrogen. The PVS2 contains(w/v) 30% glycerol, 15% ethylene glycol and 15% DMSO in Murashige-Tucker medium(MT) containing 0.15 M sucrose. Cells were treated with 60% PVS2 at 25°C for 5 min and then chilled PVS2 at 0°C for 3 min. The cell suspension of about 0.1 ml was loaded in a 0.5 ml transparent plastic straw and directly plunged in liquid nitrogen for 30 min. After rapid warming, the cell suspension was expelled in 2 ml of MT medium containing 1.2 M sucrose. The average rate of survival was about 80%. The vitrified cells regenerated plantlets. This method is very simple and the time required for cryopreservation is only about 10 min.Abbreviations DMSO dimethyl sulfoxide - PVS2 vitrification solution - LN liquid nitrogen - DSC differential scanning calorimeter - BA 6-benzylaminopurine - MT Murashige-Tucker basal medium - INAA naphthaleneacetic acid