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 human endothelial cells for vascular tissue engineering

To investigate the influence of cryopreservation on endothelial cell growth, morphology, and function human umbilical vein endothelial cells (HUVECs) were frozen following a standard protocol. Cell suspensions were exposed to 10% dimethyl sulfoxide in a high-potassium solution, cooled to -80 degrees C at 1 degrees C/min and stored in liquid nitrogen for 7-36 days. Samples were thawed in a 37 degrees C water bath and the cryoprotectant was removed by serial dilution. The growth of cell suspensions was assayed by culturing 7300 cells/cm2 for 3-5 days in order to determine the cell multiplication factor. Fresh and cryopreserved/thawed cells were analyzed for their growth, and their anti-inflammatory and anti-coagulant function by using cellular ELISA. Cryopreservation resulted in a retrieval of 66 +/- 5% and a viability of 79 +/- 3%. Cryopreserved/thawed and fresh cells showed identical doubling times and identical cell counts in the confluent monolayers. However, the lag phase of thawed HUVECs was approximately 36 h longer, resulting in significant differences in the cell multiplication factor at 3 and 5 days after seeding. After expansion to a sufficient cell count the lag phases were identical. Fresh and cryopreserved/thawed cells showed comparable anti-inflammatory and anti-coagulant activity, as judged by the basal and TNF-induced VCAM-1, ICAM-1, E-selectin, and thrombomodulin expression. Cryopreserved/thawed and recultivated endothelial cells are suitable for endothelialization of autologous allograft veins. Such tissue-engineered grafts will offer the necessary clinical safety for those patients who lack autologous material.     

Cryopreservation of bone marrow for the autologous transplantation in children

In preparing the autologous transplantation of children a method for cryoconservation of bone-marrow was developed by means of investigating the donor's bone-marrow. This method is adapted to our conditions, can easily be practised and is cell-preserving. Quantity and quality of the stored bone-marrow cells were evaluated concerning their proliferation capability by means of CFU-c assays. The highest recovery in CFU-c (78%) and cells (98%) was observed if isolated mononuclear cells with cryoprotective addition of 5% DMSO, 20% of human albumin, and 20% of serum were slowly frozen at a controllable rate, stored in liquid oxygen and thawed very quickly. According to the elaborated method the remission marrow was taken from 15 children affected with malignant diseases for autologous reinfusion. The data gained here confirm the experimental experiences.     

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 human heart cells

Single cells were isolated from human heart specimens and preserved at -56 degrees C in a cryoprotecting solution containing dimethyl sulfoxide. The cells were reanimated by rapid thawing and the properties of the cells were evaluated in a physiological solution. The reanimated cells showed morphological and physiological properties similar to those seen in normal, freshly isolated cells. These results demonstrate the feasibility of storing human heart cells for long-term studies.     

Cryopreservation of marrow, purging and autologous bone marrow transplantation in childhood

Since 1984 bone marrow from 42 children with acute lymphoblastic leukaemia, non Hodgkin's lymphoma and neuroblastoma was cryopreserved. In 5 cases (c-ALL, NHL and B type) the marrow was purged by using a cocktail of three monoclonal antibodies (VIL A1, VIB C5, VIB-E3). Up to now 13 children (ALL/10, neuroblastoma/3) were autografted (one of them after purging) after supralethal chemoradiotherapy. Except one child with early death all patients had engraftment: a level of 1.0.10(9)/l leukocytes was reached at days 10-33 (median, 19); platelet level over 60.10(9)/l at days 32-60 (median, 41). 2 children died on treatment related complications, one on infection after full haematological restitution, 2 patients alive with relapse, 8/13 alive in CCR and well.     

Mesenchymal cells from human amniotic fluid survive and migrate after transplantation into adult rat brain

Amniotic fluid has been recently suggested as an alternative source of mesenchymal stem cells. However, the fate of amniotic fluid-derived mesenchymal stem cells (AF-MSCs) after in vivo transplantation has yet to be determined. In the present study we explored whether human AF-MSCs could survive and migrate following transplantation into the striatum of normal and ischemic rat. We found that the grafted cells could survive and migrate towards multiple brain regions in the normal animals, while they moved towards the injured region in the ischemic rat. Double-immunostaining analyses showed that the implanted human AF-MSCs express markers for immature neurons (Doublecortin) at 10 days, and for astrocytes (GFAP) at 10, 30 and 90 after transplantation. This study provides the first evidence that human amniotic fluid contains cells having the potential to survive and integrate into adult rat brain tissue and, therefore, to function as effective stem cells for therapeutic strategies.     

Neural stem cells in the adult human brain

New neurons are continuously generated in certain regions of the adult brain. Studies in rodents have shown that new neurons are generated from self-renewing multipotent neural stem cells. Here we demonstrate that both the lateral ventricle wall and the hippocampus of the adult human brain harbor self-renewing cells capable of generating neurons, astrocytes, and oligodendrocytes in vitro, i.e., bona fide neural stem cells.     

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.     

Cryopreservation of isolated human hepatocytes for transplantation: State of the art

Hepatocytes isolated from unused donor livers are being used for transplantation in patients with acute liver failure and liver-based metabolic defects. As large numbers of hepatocytes can be prepared from a single liver and hepatocytes need to be available for emergency and repeated treatment of patients it is essential to be able to cryopreserve and store cells with good thawed cell function. This review considers the current status of cryopreservation of human hepatocytes discussing the different stages involved in the process. These include pre-treatment of cells, freezing solution, cryoprotectants and freezing and thawing protocols. There are detrimental effects of cryopreservation on hepatocyte structure and metabolic function, including cell attachment, which is important to the engraftment of transplanted cells in the liver. Cryopreserved human hepatocytes have been successfully used in clinical transplantation, with evidence of replacement of missing function. Further optimisation of hepatocyte cryopreservation protocols is important for their use in hepatocyte transplantation.     

Serum after autologous transplantation stimulates proliferation and expansion of human hematopoietic progenitor cells

Regeneration after hematopoietic stem cell transplantation (HSCT) depends on enormous activation of the stem cell pool. So far, it is hardly understood how these cells are recruited into proliferation and self-renewal. In this study, we have addressed the question if systemically released factors are involved in activation of hematopoietic stem and progenitor cells (HPC) after autologous HSCT. Serum was taken from patients before chemotherapy, during neutropenia and after hematopoietic recovery. Subsequently, it was used as supplement for in vitro culture of CD34(+) cord blood HPC. Serum taken under hematopoietic stress (4 to 11 days after HSCT) significantly enhanced proliferation, maintained primitive immunophenotype (CD34(+), CD133(+), CD45(-)) for more cell divisions and increased colony forming units (CFU) as well as the number of cobblestone area-forming cells (CAFC). The stimulatory effect decays to normal levels after hematopoietic recovery (more than 2 weeks after HSCT). Chemokine profiling revealed a decline of several growth-factors during neutropenia, including platelet-derived growth factors PDGF-AA, PDGF-AB and PDGF-BB, whereas expression of monocyte chemotactic protein-1 (MCP-1) increased. These results demonstrate that systemically released factors play an important role for stimulation of hematopoietic regeneration after autologous HSCT. This feedback mechanism opens new perspectives for in vivo stimulation of the stem cell pool.     

Construction of viable mouse-human hybrid cells by nuclear transplantation

Viable interspecies cytoplasmic-nuclear hybrid cells were constructed by fusion of karyoplasts prepared from the highly tumorigenic A9 mouse fibroblast cell line and cytoplasts prepared from the Detroit 532 normal human diploid cell strain. The identity of the hybrid cells was ascertained using a variety of morphological, immunological, and genetic criteria, including: nuclear pattern of staining with the fluorochrome Hoechst 33258, appearance of the actin-myosin containing cytoskeleton, presence of fibronectin, and resistance to azaguanine and diphtheria toxin. About 90% of the hybrid cells were viable, that is, capable of division. Changes in the morphology of the hybrid cells, apparently nuclear directed, were observed before cell division occurred. Using the techniques described here, large numbers of interspecies hybrid cells suitable for many types of biochemical analyses can be routinely produced.     

Cryopreservation of human cadaveric bone marrow cells

The viability of cryopreserved human cadaveric bone marrow cells was studied using methylcellulose clonal cell culture assays. This is the first study done to reveal the persistence of hemopoietic proliferative and differentiative functions using the methylcellulose clonal cell assay in cryopreserved human cadaveric bone marrow cells which are several hours postmortem. Studies of cryopreserved human cadaveric bone marrows corresponded with those of murine bone marrows. These results strongly suggest that several hours postmortem human cadaveric bone marrow cells can be cryopreserved and may be useful for transplantation.     

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.