Cryopreservation of adipose tissue

The main obstacle to achieving favorable outcome of soft-tissue augmentation after autologous fat transplantation is unpredictable long-term results due to the high rate of absorption in the grafted site. At the present time, adipose aspirates can only be used for immediate autologous fat grafting during the same procedure in which liposuction is performed; therefore adipose aspirates obtained from the procedure are usually discarded. it has been a strong desire of both surgeons and patients to be able to preserve the adipose aspirates, if an optimal technique were available, for potential future applications. For the last several years, cryopreservation of adipose tissue has been studied extensively in the author''s laboratory. Several findings from this exciting translational research will lead to develop a reliable method for long-term preservation of adipose tissue in the future. in addition, successful long-term preservation of adipose tissue may open a new era in adipose tissue related tissue regeneration.     

Cryopreservation of fetal rat brain tissue later used for intracerebral transplantation

Intracerebral grafting of immature brain tissue is now widely used as a tool to study neuronal development and regeneration in the brain and spinal cord. This has stimulated the interest in methods for storage of such tissue before transplantation. In this study a method for cryopreservation of immature rat central nervous tissue is presented and discussed in relation to current cryobiological principles. The method was applied to brain tissue from 16- and 17-day-old fetal rats, including the neocortex, habenula, septum and basal forebrain, cerebellum, and retina. After storage in liquid nitrogen from 6 to 52 days the tissue was grafted into the brain of adult rats. The recipients survived for 23 to 673 days before their brains were processed by current neuroanatomical, histological methods. The presence of graft tissues was recorded and their cellular and connective organization was examined, including their exchange of nerve connections with the host brain. The results obtained were comparable with results from other studies where the same tissues were grafted immediately after removal from the donor, and a study of cryopreservation of developing hippocampal tissue. We conclude that cryopreservation is a reliable method for storage of immature neural tissue later to be used for intracerebral grafting.     

Cryopreservation of adipose tissue

The main obstacle to achieving favorable outcome of soft-tissue augmentation after autologous fat transplantation is unpredictable long-term results due to the high rate of absorption in the grafted site. At the present time, adipose aspirates can only be used for immediate autologous fat grafting during the same procedure in which liposuction is performed; therefore adipose aspirates obtained from the procedure are usually discarded. It has been a strong desire of both surgeons and patients to be able to preserve the adipose aspirates, if an optimal technique were available, for potential future applications. For the last several years, cryopreservation of adipose tissue has been studied extensively in the author’s laboratory. Several findings from this exciting translational research will lead to develop a reliable method for long-term preservation of adipose tissue in the future. In addition, successful long-term preservation of adipose tissue may open a new era in adipose tissue related tissue regeneration.     

Cryopreservation of brain tissue for primary culture.

Factors affecting recovery of brain cells from cryopreserved cerebral tissues of fetal rats were examined based on yields of viable cells on cell culture. Favorable preservation was obtained with freezing small pieces (less than 1 mm cube) of brain tissues rather than whole tissues or dissociated single cells, and use of 10% dimethylsulfoxide as a cryoprotectant in liquid nitrogen. As for cell preparation procedures, cell survival was improved when tissues were heated at 32 degrees C during papain digestion and centrifugation. Under favorable conditions, the number of brain cells recovered from cryopreserved tissues corresponded to 20-30% of those from fresh control tissues. Immunocytochemical characteristics of cultured neurons, astrocytes, and oligodendrocytes from cryopreserved and fresh tissues were indistinguishable. Semi-quantitive analyses of microtubule-associated protein-2 (MAP-2) and synaptophysin revealed that there was no difference in the amounts of these markers between cultures from both fresh and cryopreserved tissues. These results suggest that most of all cell types including neurons were equally susceptible to the cryopreservation procedures. We concluded that cryopreservation in liquid nitrogen is an effective method for preservation of embryonic brain tissues for later use in cell culture studies.     

Cryopreservation of adherent human embryonic stem cells

Standard human embryonic stem (HES) cell cryopreservation methodologies, including slow freezing and vitrification of colonies in suspension, are plagued by poor viability and high differentiation rates upon recovery. To facilitate research studies and clinical applications of HES cells, we have developed a cryopreservation technique based on stabilizing HES colonies adherent to or embedded in a Matrigel matrix. This method increases cell viability by over an order of magnitude compared with cryopreservation in suspension and reduces differentiation. Loading adherent HES cells with the disaccharide trehalose prior to cryopreserving in a dimethylsulfoxide-containing cryoprotectant solution further improves cell viability under certain conditions. Our proposed approach has the potential to reduce the time required to amplify frozen stocks of HES cells, minimize risk of clonal selection during freeze-thaw cycles, and facilitate storage of HES cell clone libraries.     

Cryopreservation of somatic embryos and embryonic axes of Camellia japonica L.

Summary Cryopreservation in liquid nitrogen was attempted with both somatic embryos and zygotic embryonic axes of the ornamental Camellia japonica L. Several protective measures were applied to somatic embryos (desiccation, chemical protectors, hardening by culture at low temperatures, encapsulation in alginate beads), but none allowed somatic embryos cultures to survive after 24 h in liquid nitrogen. Embryonic axes, however, were easily cryopreserved by means of the simplest technique: desiccation in a laminar flow hood and direct immersion in liquid nitrogen. Although the causes of the difference in cryopreservability between the two types of material are not known, one might be the difference between their degrees of differentiation and water content.Abbreviations ANOVA analysis of variance - BA N6-benzyladenine - DMSO dimethyl sulfoxide - IBA indole-3-butyric acid - LN liquid nitrogen - MS Murashige and Skoog mineral solution - fwt fresh weight - LSD Least Significant Difference     

精神依赖物海洛因对大鼠胚胎发育和脑组织Bax表达的影响

目的 探讨精神依赖物海洛因对胚胎大鼠发育和大脑Bax表达的影响。方法 将受孕后的Wistar大鼠随机分为对照组和海洛因给药组(分为海洛因低、中、高剂量组)。第7天,分别给予16、32和64mg/kg的海洛因,连续给予海洛因9d,观察精神依赖物海洛因对胚胎大鼠形态结构发育的影响,用酶联免疫吸附法(ELISA)检测胚胎大脑组织Bax表达水平。结果 胚胎观察发现,海洛因低、中、高剂量组活胚胎总数比对照组分别减少了27.27%、37.12%和48.48%;海洛因低剂量组胚胎枕骨发育不全,胚胎出现脑膨出畸形;海洛因中剂量组胚胎枕骨、顶骨发育不全,胚胎脑膨出明显;海洛因高剂量组胚胎枕骨、顶骨、颞骨发育不全,胚胎脑膨出更为明显。ELISA检测发现,海洛因低、中、高剂量组胚胎大脑组织中Bax表达水平比对照组分别增加了11.41%、47.06%、83.74%,差异有显著性(P<0.05,P<0.01);胚胎小脑组织中Bax表达水平比对照组增加了17.16%、52.96%和90.01%,差异有显著性(P<0.05,P<0.01)。结论 精神依赖物海洛因具有明显抑制胚胎大鼠形态结构发育的作用,抑制作用随给予海洛因剂量的增加而增强,其作用机制可能与海洛因诱导胚胎组织器官Bax表达上调有关。     

Cryopreservation of Quercus faginea embryonic axes

Embryonic axes, excised or included in cotyledonary tissue, of Quercus faginea were tested for viability after rapid freezing and storage in liquid nitrogen (−196 °C). Explants were previously pretreated by desiccation at different moisture contents or by soaking in cryoprotectant (15% dimethyl sulfoxide). Best germination response after freezing (60%) was observed when embryonic axes were desiccated from 53 to 21% moisture content (on a fresh weight basis). Desiccation below 39% moisture content or freezing produced damage resulting in loss of organized growth and the induction of callus formation.     

Effects of astroglia on the development of cultured neurons from embryonic rat cerebral cortex.

1. Previous studies from our laboratory demonstrated that subcultured astroglia enhance neurite outgrowth and survival of cultured neurons from embryonic rat cerebral cortex, but suppress proliferation of neuroblasts. 2. In the present study, the mechanisms of these three effects were further investigated. 3. Dissociated neurons were seeded on poly-L-lysine-coated coverslips which were plated on subcultured astroglia, and the survival, proliferation and neurite outgrowth of the neurons were investigated. Under these conditions, survival and antimitotic effects were also observed, while neurite extension was not stimulated. 4. The results clearly indicate that neuronal survival and proliferation are regulated by soluble factors produced by astroglia. 5. We also postulated that the neurite-promoting effect of astroglia is mediated by cell-cell contact. 6. This idea was confirmed by the finding that neurite extension was enhanced when the neurons were cultured directly on heat-treated astroglia. 7. The neurite-promoting effect was found to be specific to astroglia. 8. We preliminarily characterized the astroglial surface neurite-promoting factors (ASNPFs). 9. The relationship of laminin to ASNPFs was examined by using antibody to laminin. Laminin antibody did not inhibit the ASNPF activity. 10. The effect of digestion of heat-treated astroglia with enzymes (sialidase and endo-beta-galactosidase) on the ASNPF activity was also examined. 11. These enzyme treatments did not inhibit the ASNPF activity. 12. These results suggest that enhancement of the neurite-promoting activity is not associated with the sugar moiety of ASNPFs.     

Myelin formation in dissociated cell cultures of rat embryonic cerebral hemispheres

Developing cultures of dissociated cerebral hemispheres obtained from 18-day-old embryonic rats synthesized, or activated, a myelin-related enzyme, 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNPase). This increase in activity coincided with the onset of myelination. The presence of CNPase in oligodendrocytes and myelin was demonstrated using immunocytochemical staining techniques. Myelinated axons and myelin sheaths were clearly made visible by electron microscopy of 28-day-old cultures.     

Cryopreservation of oocyte and ovarian tissue

Cryopreservation of reproductive cells (i.e., oocytes, spermatozoa) and tissues (i.e., ovarian and testicular tissue) is a developing technology that has tremendous implications for rapid advancement of biomedical research in general. Since the early 1980s, advances have been made in establishing optimal conditions for in vitro oocyte maturation, fertilization, and culture of resulting embryos. These in vitro systems have contributed significantly to the utilization of these cells and tissues after thawing and have made it possible to evaluate protocols designed to cryopreserve such biomaterials more effectively. Although cryopreservation of preimplantation embryos from various species including mouse, human, and farm animals has been successful, cryopreservation of oocytes from most mammalian species has been more challenging due to their extreme sensitivity to suboptimal conditions during the cryopreservation process. Cryopreservation on mouse oocytes have been well documented and have resulted in greater success than studies with other mammalian species. Ovarian tissue cryopreservation and transplantation techniques have recently received much scientific and public attention due to their great potential use in human infertility treatment, in safeguarding the reproductive potential of the endangered species, and in genome banking of genetically important lab animal strains. A review of past and current research in the field of oocyte and ovarian tissue cryopreservation and transplantation and discussion of possible strategies for oocyte and ovarian tissue banking are provided.     

Cryopreservation of isolated rat hepatocytes

Summary Isolated parenchymal hepatocytes from adult rats were frozen in media containing 10% glycerol, 10% dimethylsulfoxide (DMSO), or 20% DMSO. Three microsome-associated functions were compared in nonfrozen cells and cells frozen in each of the above cryoprotectant solutions. Freezing in DMSO maintains cytochromes P-450 and b₅ and NADPH-cytochrome C reductase at levels nearer to control values than does freezing in glycerol. Cells frozen and subsequently thawed and cultured for 24 h lose a greater amount of cytochrome P-450 than do nonfrozen cultured cells. The levels of cytochrome b₅ and reductase in frozen-thawed cells remain close to control values. Cell viability (trypan blue dye exclusion and percentage of attached cells) after freezing is maintained better using DMSO as a cryoprotectant. Dimethylsulfoxide protects the hepatocytes from freeze-induced damage to the extent that many viable cells attach to collagen-coated petri dishes, survive for at least 24 h, and still maintain significant levels of enzymes of importance to drug and carcinogen metabolism. This work was supported by Grant CA-30241 from the National Institutes of Health, Bethesda, Maryland.     

Cryopreservation and quality control of mouse embryonic feeder cells

Stem cell research is a highly promising and rapidly progressing field inside regenerative medicine. Embryonic stem cells (ESCs), reprogrammed “induced pluripotent” cells (iPS), or lately protein induced pluripotent cells (piPS) share one inevitable factor: mouse embryonic feeder cells (MEFs), which are commonly used for ESC long term culture procedures and colony regeneration. These MEFs originate from different mouse strains, are inactivated by different methods and are differently cryopreserved. Incomprehensibly, there are to date no established quality control parameters for MEFs to insure consistency of ESC experiments and culture. Hence, in this work, we developed a bench-top quality control for embryonic feeder cells.According to our findings, MEFs should be inactivated by irradiation (30 Gy) and cryopreserved with optimal 10% DMSO at 1 K/min freezing velocity. Thawed cells should be free of mycoplasma and should have above 85 ± 13.1% viability. Values for the metabolic activity should be above 150 ± 10.5% and for the combined gene expression of selected marker genes above 225 ± 43.8% compared to non-irradiated, cryopreserved controls. Cells matching these criteria can be utilized for at least 12 days for ESC culture without detaching from the culture dish or disruption of the cell layer.     

Morphological differentiation of embryonic rat sympathetic neurons in tissue culture. II. Serum promotes dendritic growth

In the preceding paper, we reported that embryonic rat sympathetic neurons formed axons, but not dendrites, when they were maintained in the absence of serum and nonneuronal cells. To assess the effects of serum-derived factors on cellular morphology, cultures were initially maintained in serum-free medium while nonneuronal cells were eliminated. Subsequently some cultures were chronically exposed either to fetal calf serum (10%) or to a high-molecular-weight ammonium sulfate fraction of serum (P40 material, 500 micrograms/ml). Phase-contrast microscopy revealed that serum and P40 material did not alter neuronal survival, but did cause flattening of the somata and fasciculation of processes. When neurons exposed to serum or P40 material were injected with Lucifer Yellow, it was found that the majority (greater than or equal to 90%) had local, tapered processes that could be identified as dendrites by light microscopic criteria. These local processes also exhibited other dendritic characteristics in that (1) they reacted with monoclonal antibodies to nonphosphorylated forms of the M and H neurofilament subunits and to microtubule-associated protein 2; and (2) they had substantial amounts of RNA as determined by [3H]uridine autoradiography. Quantitative measurements of the effects of serum and P40 material on dendritic morphology revealed that (1) an 8-day exposure caused most neurons (greater than 80%) to form dendrites; (2) neurons typically had more than one dendrite (mean of 4.1 +/- 0.2 dendrites/cell after a 28-day exposure); and (3) the dendrites were relatively short with the maximum extent of the dendritic arbor being 110 +/- 13 micron after 4 weeks. Serum and P40 material did not routinely cause the formation of supernumerary axons, did not alter radial axonal outgrowth from ganglion explants, and did not significantly increase [3H]leucine incorporation. Thus, serum contains a factor (or factors) which selectively stimulates the extension of dendrites, but not axons. If such a factor were operative in situ, it could play an important role in determining the morphology of sympathetic neurons. In examining the mechanism of serum-induced dendritic growth, we found that even high concentrations (5 micrograms/ml) of nerve growth factor failed to promote dendritic growth in the absence of serum; thus, nerve growth factor by itself is not a sufficient condition for the extension of dendrites.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cryopreservation of rat blastocysts by vitrification

Rat blastocysts equilibrated with vitrification solution (VS1), consisting of dimethyl sulfoxide, acetamide, propylene glycol, and polyethylene glycol were plunged directly into liquid nitrogen. The embryo suspension are solidified by an extreme elevation in viscosity of solution. The embryos are cryopreserved by vitrification without intra- and extracellular ice formation. The proportion of morphologically normal embryos after cooling and warming was 79% (117/149) and all (48/48) of the embryos cultured were developed to expanded or hatched blastocysts. Normal live young were obtained 41% of the time (28/69) after transfer of the cooled and warmed embryos to pseudopregnant recipients.     

Cryopreservation of erythropoietin-responsive cells in murine hematopoietic tissue

The optimal conditions were determined under which maximum survival of murine hematopoietic erythropoietin-responsive cells (ERC) could be ensured during manipulations required for cryopreservation. Cell survival was similar over freezing rates between 2 and 10 °C/min. Optimal cryoprotectants were 10% dimethyl sulfoxide (DMSO) and 20% fetal calf serum; the DMSO was removed by centrifugation after stepwise dilution with 20 vol of medium over a 10-min period. Differing thawing rates for the cell suspensions had minimal effects on survival. “Seeding” the cell suspensions with ice crystals had no effect on ERC recovery. Overall ERC survival varied between 20 and 40%. These results confirm earlier reports that certain ERC populations are more sensitive to damage during cryopreservation than are other hematopoietic progenitor cells.