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.     

不同降温速率对脐血干细胞冷冻复苏后生物学特性的影响

考察了不同降温速率对脐血造血干细胞各种生物学特性的影响。在4℃～-40℃的降温范围内,分别选择-0.5℃/min, -1℃/min, -5℃/min的降温速率进行降温,对复苏后的脐血单个核细胞的回收率、活性和CD34+含量的变化以及BFU-E、CFUGM和CFU-MK集落的回收率进行了考察,发现在-1℃/min的降温速率下,脐血MNC回收率可达93.3%±1.8%,活性可达95.0%±3.9%, CD34^＋细胞回收率达80.0%±17.9%,BFUE回收率为87.1%±5.5%,CFUGM回收率达88.5%±8.9%,CFUMK的回收率也达到86.2%±7.4%。并且对复苏后的细胞进一步进行体外培养,发现在-1℃/min的降温速率下复苏的细胞仍然具有与未经冷冻细胞相似的扩增能力,而-0.5℃/min和-5℃/min这两种降温速率条件下复苏的细胞与未经冷冻的细胞相比差距较大。因而-1℃/min的降温速率对冻存脐血干细胞比较合适。     

A technical bias: differences in cooling rates prevent ampoules from being a reliable index of stem cell cryopreservation in large volumes

Ampoule tests are commonly used as an index of the cryopreservation efficiency of marrow stem cells in bags. We have studied the recovery of hematopoietic progenitor cells (CFU-GM, BFUe) in 52 ampoules and compared it to the recovery in 83 standard bags. Our data showed significantly deficient CFU-GM and BFUe recoveries (respectively 47 +/- 31% and 31 +/- 30%) in ampoules when compared to bags (respectively 72 +/- 22% and 64 +/- 19%; P less than 0.001). Moreover, a good progenitor cell recovery (greater than or equal to 50%) was observed in only 46% of frozen ampoules versus 100% observed in frozen bags (P less than 0.05). We were able to relate this nonoptimal recovery to an excessively rapid freezing rate of -9 degrees C/min following the release of fusion heat which occurred in ampoules, while the freezing rate was constantly maintained at -2 degrees C/min in the corresponding bags. We therefore conclude that the cooling conditions have to be carefully controlled to ensure that the bags and ampoules are both cooled under the same conditions. Otherwise, ampoules would not be a reliable index of the true progenitor cells' cryopreservation efficiency in bags.     

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.     

Repopulating potential of hematopoietic precursor cells

Experiments were performed in 1800 cGy whole-body x-irradiated dogs. Mononuclear cells were collected from bone marrow, peripheral blood, and fetal liver. They were cryopreserved in -196 degrees C liquid nitrogen until used for transplantation. The thawed transfusates were adjusted to contain 1.5-1.6 x 10(5) CFU-GM per kg body weight. The blood granulocyte recovery was rapid after transfusion of blood-derived stem cells as compared to the use of bone-marrow-derived stem cells. In both instances, however, normal values were not reached for several weeks. In contrast, the use of fetal-liver-derived stem cells resulted in a very rapid initial granulocyte increase with a return of values to normal (or even overshoot) within 3 weeks after transplantation. A biomathematical granulocyte renewal simulation system is described that permits calculation of the absolute number of pluripotent stem cells in the transfusate. The data after fetal liver stem cell transplantation can be fitted only if an initial stem cell replication rate of 0.95 is assumed (in contrast to 0.65 using bone marrow or blood-derived stem cells).     

Conservation of bone marrow CFUc in different phases of the cell cycle during cryopreservation

The effect of low temperature (-196 degrees C) preservation on the recovery of colon-forming units (CFUs) of bone marrow at different phases of the cell cycle before cryopreservation is dealt with. The intact bone marrow "enriched" with CFUs in S phase of the cell cycle and the bone marrow without colony-forming units in S phase were exposed to cryopreservation. After cryopreservation of the bone marrow enriched with CFUs in S phase and th bone marrow without colony-forming units in S phase the number of CFUs decreases by the same value as in the cryopreserved bone marrow obtained from intact mice.     

Multiple classes of prostaglandin F2 alpha binding sites in subpopulations of ovine luteal cells

A cryostorage procedure was developed to provide ovine luteal cells throughout the period of seasonal anestrus. Corpora lutea obtained from midluteal phase, superovulated ewes were dispersed enzymatically. Some dispersed cells were fractionated into subpopulations by elutriation. Dimethylsulfoxide (7.5% final concentration) in Hanks' buffered saline was added to cells at 4 degrees C, and dispersed cell preparations were frozen in a programmable cell freezer and stored at -196 degrees C. After recovery from cryopreservation, cell viability and prostaglandin F2 alpha (PGF2 alpha) binding characteristics of thawed cells were not different from those of corresponding fresh cells. Additionally, thawed cells retained the capacity to attach to culture dishes and retained responsiveness of progesterone secretion to prostaglandin E2 (PGE2) and ovine luteinizing hormone (LH), although rates of progesterone secretion were attenuated in thawed compared with fresh cells. The cryopreservation procedure will prove useful to relieve constraints in utilization of ovine luteal cells arising from reproductive seasonality in sheep. Cells retrieved from cryostorage were evaluated by studying PGF2 alpha binding characteristics. From saturation analyses (increasing amounts of radiolabeled PGF2 alpha) of PGF2 alpha binding to unfractionated cells, we detected a single class of high affinity binding sites (Kd = 17.4 +/- 2.3 nM) in addition to the nonspecific binding component. Using displacement analyses (constant radiolabeled PGF2 alpha and increasing amounts of unlabeled PGF2 alpha) and unfractionated cells, we detected additional binding sites of lower affinity (Kd = 409 +/- 166 nM) as well as the nonspecific binding component. Small luteal cells obtained by elutriation, which were essentially devoid of large cell contamination, had only low affinity binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Effect of different freezing rates during cryopreservation of rat mesenchymal stem cells using combinations of hydroxyethyl starch and dimethylsulfoxide

ABSTRACT: BACKGROUND: Mesenchymal stem cells (MSCs) are increasingly used as therapeutic agents as well as research tools in regenerative medicine. Development of technologies which allow storing and banking of MSC with minimal loss of cell viability, differentiation capacity, and function is required for clinical and research applications. Cryopreservation is the most effective way to preserve cells long term, but it involves potentially cytotoxic compounds and processing steps. Here, we investigate the effect of decreasing dimethyl sulfoxide (DMSO) concentrations in cryosolution by substituting with hydroxyethyl starch (HES) of different molecular weights using different freezing rates. Post-thaw viability, phenotype and osteogenic differentiation capacity of MSCs were analysed. RESULTS: The study confirms that, for rat MSC, cryopreservation effects need to be assessed some time after, rather than immediately after thawing. MSCs cryopreserved with HES maintain their characteristic cell surface marker expression as well as the osteogenic, adipogenic and chondrogenic differentiation potential. HES alone does not provide sufficient cryoprotection for rat MSCs, but provides good cryoprotection in combination with DMSO, permitting the DMSO content to be reduced to 5%. There are indications that such a combination would seem useful not just for the clinical disadvantages of DMSO but also based on a tendency for reduced osteogenic differentiation capacity of rat MSC cryopreserved with high DMSO concentration. HES molecular weight appears to play only a minor role in its capacity to act as a cryopreservation solution for MSC. The use of a 'straight freeze' protocol is no less effective in maintaining post-thaw viability of MSC compared to controlled rate freezing methods. CONCLUSION: A 5% DMSO / 5% HES solution cryopreservation solution using a 'straight freeze' approach can be recommended for rat MSC.     

Bone marrow cryopreservation and clinical implications in autologous bone marrow transplantation

A simple, rapid and effective technique using the IBM (Cobe)-2991 cell processor for the concentration of buffy coat cells from large volume marrow has been well adopted (n = 16). Only about one-eighth of the original volume was obtained while retaining more than 90% of the total nucleated cells to be cryopreserved in polyolefine bags with TC-199 culture medium and final 10% dimethylsulfoxide (DMSO) (n = 9), processed by a computerized Nicool ST-20 (France) programmed freezer and stored in a vapor phase of liquid nitrogen at -196 degrees C. Stem cell assay by CFU-GM after thawing yielded a mean of 50.39 +/- 19.54% which has been satisfactory for clinical implementation. So far, three cases with hematological malignancies had been rescued by autologous cryopreserved marrow after supralethal doses of chemoradiotherapy. Two patients with acute nonlymphocytic leukemia transplanted in 1st remission as of Oct. 31 had been disease free for 178+ and 157+ days, respectively, after transplant which was taken at the corresponding age of 53 and 42 years. The other patient who was a victim of Hodgkin's disease, stage IV, and was transplanted in 3rd remission, expired on the 59th day because of the complication of idiopathic interstitial pneumonitis despite excellent granulocytopoietic reconstitution. The preliminary results are encouraging for further exploitation, especially for those who would otherwise be candidates for allogeneic bone marrow transplantation but are limited by age or lack of an HLA-identical sibling to serve as marrow donors.     

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。     

Proliferation, multipotency and neuronal differentiation of cryopreserved neural progenitor cells derived from the olfactory neuroepithelium of the adult rat

The use of olfactory neuroepithelium neural progenitor cells for transplantation has attracted attention in the treatment of many neurological disorders, which require efficient recovery methods and cryopreservation procedures. The purpose of this study was to evaluate different cryopreservation techniques for neural progenitor cells derived from the olfactory neuroepithelium (ONe NPCs) in adult rats. Initially, we compared the survival rates of cryopreserved ONe NPCs treated with six different cryoprotectants: dimethylsulfoxide (DMSO), ethylene glycol (EG) and glycerol, each with or without 10% FBS and with two different storage periods in liquid nitrogen (-196 degrees C), specifically 3 days short-term storage and 3 months long-term storage. We assessed the recovery efficiency of ONe NPCs after freezing and thawing by viability testing and colony-forming assay as well as immunocytochemistry under different conditions. No significant difference in the survival rate was observed among these different cryoprotectants. With these protocols, ONe NPCs retained their multipotency and differentiated into glial (GFAP-positive), neuronal (NeuN-positive) and oligodendroglia (Galc-positive) cells. Collectively, our results imply that, under optimal conditions, ONe NPCs might be cryopreserved for periods of >3 months without losing their proliferative and multipotency activities.     

Synergistic effects of liposomes, trehalose, and hydroxyethyl starch for cryopreservation of human erythrocytes

Red blood cells (RBCs) can be cryopreserved using glycerol as a cryoprotective agent, but one of the main disadvantages is the time-consuming deglycerolization step. Novel cryopreservation strategies for RBCs using nontoxic cryoprotective agents are urgently needed. The effect of DMPC, DOPC, and DPPC liposomes on survival of RBCs cryopreserved with trehalose and HES has been evaluated. DMPC caused hemolysis before freezing and affected RBC deformability parameters. DMPC treated RBCs displayed a strong increase in trehalose uptake compared to control cells, whereas DOPC treated liposomes only displayed a slight increase in trehalose uptake. High intracellular trehalose contents were observed after cryopreservation. The recovery of cells incubated with trehalose and liposomes, frozen in HES ranged between 92.6 and 97.4% immediately after freezing. Recovery values of RBCs frozen in HES, however, decreased to 66.5% after 96 h at 4°C compared to 77.5% for DOPC treated RBCs. The recovery of RBCs incubated and frozen in trehalose medium was 77.8%. After 96 hours post-thaw storage recovery of these cells was 81.6%. DOPC and DPPC treated RBCs displayed higher recovery rates (up to 89.7%) after cryopreservation in trehalose compared to control RBCs. Highest survival rates were obtained using a combination of trehalose and HES: 97.8% directly after thawing and 81.8% 96-h post-thaw. DOPC liposomes, trehalose and HES protect RBCs during cryopreservation in a synergistic manner. The advantage is that the protective compounds do not need to be removed before transfusion.     

Variation of the HES concentration for the cryopreservation of keratinocytes in suspensions and in monolayers

It has recently been shown that keratinocytes, both in suspension and in monolayers, can be successfully cryopreserved with hydroxyethyl starch (HES) (6, 9). HES is a nontoxic biodegradable macromolecule which is clinically approved as a plasma expander and which has already been used for the cryopreservation of red blood cells (10, 11). In this study we varied the HES concentration between 0 and 10 wt% in 2% steps for suspended cells and between 0, 4, 6, 8, and 10 wt% for monolayer cells in order to determine the effect on the survival rate and metabolic activity after cryopreservation. The experiments with the suspended cells were performed both with and without NCS. Cryopreserved keratinocytes can be transplanted onto patients for the treatment of deep dermal burns and leg ulcers. In this study, we achieved a survival rate of 80% for the suspended cells (10 wt% HES, 3 degrees C/min) and a survival rate of even 88% when the cells were cryopreserved as a monolayer using the same parameters. The addition of NCS did not improve the results for the suspended cells significantly.     

The effect of cryopreservation on ciliary beat frequency of human respiratory epithelium

The effect of cryopreservation on ciliary activity of human nasal respiratory epithelium was evaluated. Samples were cryopreserved in a solution containing nutrient medium, 10% fetal calf serum, and two different concentrations (10 or 20%) of dimethyl sulfoxide and stored in liquid nitrogen at -196 degrees C for 2 weeks. Ciliary beat frequencies (CBF) of the samples before and after cryopreservation were compared. Mean CBF values did not differ significantly with both concentrations of dimethyl sulfoxide. The mean intrasample coefficient of variation of the CBF decreased significantly after cryopreservation. After thawing, CBF remained unchanged for at least 4 hr. It is concluded that normal ciliated epithelial cells can be frozen and stored in liquid nitrogen at 196 degrees C while maintaining their CBF.     

Cryopreservation of viable hepatocyte monolayers in cryoprotectant media with high serum content: metabolism of testosterone and kaempherol post-cryopreservation

Little work in the literature focuses on the cryopreservation of primary hepatocytes as monolayer cultures, yet this technique offers many distinct advantages over other cryopreservation systems, including high recovery, high post-thaw nutrient penetration, and low numbers of trapped dead cells. This article investigates the cryopreservation of primary rat hepatocytes at -78 degrees C attached as monolayers to collagen coated culture dishes, and describes efforts to increase post-thaw viability and function through manipulation of the freeze/thaw protocol. Different concentrations of foetal calf serum (FCS) with 10% (v/v) dimethyl sulphoxide (ME2SO) were tested as cryopreservation media, and high cryoprotectant serum levels were found to be important in maintaining membrane integrity and function in the cryopreserved rat hepatocyte monolayer cultures. Cultures cryopreserved with 90% (v/v) FCS plus 10% (v/v) ME2SO maintain 79.7+/-6.5% of the monolayer area as viable cells with normal morphology (by image analysis), 112.7+/-14.2% protein concentration, 55.4+/-4.2% carboxyfluorescein diacetate de-acetylation, 27.2+/-7.5% kaempherol glucuronidation (a measure of UDP-glucuronosyl transferase activity), and 39.3+/-7.3% testosterone hydroxylation (a measure of cytochrome P-450 activity) compared with non-cryopreserved controls. This method of cryopreservation may provide a simple, convenient means of long-term storage of hepatocytes for in vitro metabolism studies.     

Cryopreservation of Suspension Cell Line Derived from the Protoplast Culture of Brassica campestris var. Pekinensis

Protected by DMSO, the suspension cell line derived from the protoplast culture of Brassica campestris var. pekinensis can be stored in liquid nitrogen (-196℃) for a long term. The addition of sorbitol and mannose can increase and decrease the protection, respectively. The medium also has an effect on cryopreservation. The relative survival rates of cells are little different in different days of cryopreservation. The highest rate of relative survival of cryopreserved cells reaches 75.4%. When the cryopreserved cells are thawed and resuspended, regrowth immediately occurs after just one day of lag period. Resuspended for six days, the cells increase 300–500%. It is much better for recovery of growth to resuspend in the dark than in the light. Like the non-cryopreserved control, the cryopreserved cells can be normally digested, producing a number of viable protoplasts which can be actively divided and form calli.     

Cryopreservation of cultured periosteum: effect of different cryoprotectants and pre-incubation protocols on cell viability and osteogenic potential

Evidence has accumulated that periosteal cells have a great potential to regenerate bone. We have demonstrated that cultured periosteum (CP) in membrane form is an effective device to regenerate alveolar bone. To increase the availability of CP in a clinical environment, an effective cryopreservation protocol for CP has been developed. In this study, three different cryoprotectants (Me(2)SO, glycerol, and ethylene glycol) were used. The effect on cell viability of pre-incubation temperature, pre-incubation time, and agitation during incubation was investigated. Samples were stored at -196 degrees C for 10 days. Cell viability was assessed by a colorimetric cell viability assay using a tetrazolium salt, and the assay results were confirmed by confocal laser scanning microscopy after staining with a combination of calcein AM and ethidium homodimer-1. The activity of the cells after thawing was assessed by alkaline phosphatase assay. To assess the osteogenic potential of cryopreserved CP, the CP was grafted to calvarial defects in athymic rats. The greatest cell viability was obtained in the group equilibrated at 37 degrees C for 30 min with Me(2)SO, under agitation, showing 63.3 +/- 10.5% recovery. After cryopreservation, the cell growth of surviving cells was identical when Me(2)SO was used as a cryoprotectant. Alkaline phosphatase (ALP) activity was maintained in the groups cryopreserved with Me(2)SO and glycerol. The transplantation experiment showed that the calvarial defects were completely closed by grafting cryopreserved CP, which demonstrates that the osteogenic property of CP was well maintained. An efficient cryopreservation protocol for CP has been developed and this will provide a convenient and effective treatment option for bone regeneration in clinics.     

Method for concentrating marrow stem cells using the IBM 2991 washer. Necessary preparation before in vitro treatment of bone marrow by pharmacologic or immunologic means

The technique using the IBM 2991 blood cell processor is an effective technique for the concentration of mononuclear cells from large volumes of bone marrow. The marrow cells are layered on to Ficoll Metrizoate using the IBM processing set. The mononuclear cells and CFU-GM recoveries are in close relationship with the hematocrit of the cell suspension processed. Twenty two bone marrows have been collected and purified according to this protocol. The mononuclear cell recovery is an average of 78,3% (range: 44-92%) and the CFU-GM recovery is in average of 67,5% (range: 40-89%). At the end of the procedure the cell viability is satisfying (97,1% +/- 1,7 are trypan blue negatives). When it is necessary to remove from the bone marrow collected either malignant cells prior autologous bone marrow graft or T lymphocytes in an attempt to prevent GVHD in allogeneic BMT, the purity of marrow cell suspension become a fundamental parameter.     

Biochemical functionality and recovery of hepatocytes after deep freezing storage

The present study was undertaken to define the conditions for optimal cryopreservation of hepatocytes. Two different freezing procedures were analyzed: a slow freezing rate (SFR) (-2 degrees C/min down to -30 degrees C and then quick freezing to -196 degrees C) and a fast freezing rate (FFR) (direct freezing of tubes to -196 degrees C: -39 degrees C/min). Cells were frozen in fetal bovine serum containing 10% Dimethyl sulfoxide (DMSO). After rapid thawing at 37 degrees C, followed by dilution and removal of the cryoprotectant, cells were plated and several parameters were followed as criteria for optimal cryopreservation of cells. The FFR cells showed no apparent ultrastructural damage after 24 h of culture. Plating efficiency and spreading were similar as controls. Gluconeogenesis from pyruvate and fructose, tyrosine amino transferase induction by glucagon and dexamethasone, urea production, and plasma protein synthesis of FFR cells were similar to those found in control cultures. The FFR procedure, in comparison to the SFR method, seemed to render the best preserved hepatocytes.