bluuebunny/crossref_metadata_2025 huggingface dataset:
A 9L Gliosarcoma Transplantation Model for Studying Adoptive Immunotherapy into the Brains of Conscious Rats
Authors: Fleshner Monika , Watkins Linda R. , Redd Joan M. , Kruse Carol A , Bellgrau Donald
Published: June 2017

A rat model for brain tumor immunotherapy is described that closely mimics the type of treatment that could be administered to humans. It involves surgical implantation of a permanent cannula in the brain, through which tumor cells and various effector cells and/or cytokines can be injected. The advantage of this system over more conventional animal surgical procedures is that conscious animals can be treated multiple times while avoiding morbidity and mortality associated with reoperative procedures. Using this system to study adoptive immunotherapy for brain tumors, we provide evidence that the 9L gliosarcoma tumor from the Fischer rat strain can be reduced or destroyed in situ following adoptive immunotherapy with specifically activated cytotoxic T lymphocytes.
Behavioral Effects of Neural Transplantation
Authors: Emerich Dwaine F. , Ragozzino Michael , Lehman Michael N. , Sanberg Paul R.
Published: June 2017

Considerable evidence suggests that transplantation of fetal neural tissue ameliorates the behavioral deficits observed in a variety of animal models of CNS disorders. However, it is also becoming increasingly clear that neural transplants do not necessarily produce behavioral recovery, and in some cases have either no beneficial effects, magnify existing behavioral abnormalities, or even produce a unique constellation of deficits. Regardless, studies demonstrating the successful use of neural transplants in reducing or eliminating behavioral deficits in these animal models has led directly to their clinical application in human neurodegenerative disorders such as Parkinson's disease. This review examines the beneficial and deleterious behavioral consequences of neural transplants in different animal models of human diseases, and discusses the possible mechanisms by which neural transplants might produce behavior recovery.
Myoblast Transplantation: What's Going on?
Authors: Hoffman Eric P.
Published: June 2017

Muscle tissue, due to its syncytial cellular structure and specific nerve and vascular requirements, cannot be transplanted as an organ. In an effort to use transplantation to overcome genetically determined biochemical deficiencies in muscle, attention has focused on delivering the developmental precursors of mature muscle fibers, myoblasts, as donor cells for cellular transplantation. Consequent to a brief report showing limited success of the technique in mice in 1988, human trials were summarily designed, funded, and initiated. The human trials have spurred more controversy than concrete data, with more reports appearing in the popular press than in scientific journals. This review summarizes the events leading to the current state of affairs, and underscores the biological hurdles facing myoblast transplantation before it can be considered a therapeutic modality.
Bone Marrow Transplantation: A Review
Authors: Demagalhaes-Silverman Margarida , Donnenberg Albert D. , Pincus Steven M. , Ball Edward D.
Published: June 2017

The indications for bone marrow transplantation (BMT) continue to expand as supportive care improves and alternative stem cell sources have been exploited. The application of allogeneic BMT has expanded to include unrelated histocompatibility antigen-matched donors and partially matched family donors. While the results of these transplants are not as good as those with sibling donors, these alternative donors allow curative therapy to be delivered to patients with leukemia, aplastic anemia, and immunodeficiency diseases who otherwise would not be eligible for curative therapy. Autologous BMT has emerged as a curative therapy for patients with non-Hodgkin's lymphoma, Hodgkin's disease, acute myeloid leukemia, and acute lymphoblastic leukemia. In addition, dose-intensive therapy with marrow or peripheral blood stem cell support to patients with Stage II, III, and IV breast carcinoma is under intense study in single and multiple-institution studies. Important issues under active study are prophylaxis for graft-versus-host-disease, the role of marrow purging in autologous BMT, the use of cytokine and chemotherapy-mobilized peripheral blood stem cells, and control of infectious diseases. This review summarizes current results in both allogeneic and autologous bone marrow transplantation, issues in marrow graft manipulations, issues in infectious disease control, the application of gene therapy to correct genetic disease through bone marrow or peripheral blood infusion, and current concepts in post-BMT immunization.
Co-Transplantation of Plasmid-Transfected Myoblasts and Myotubes into Rat Brains Enables High Levels of Gene Expression Long-Term
Authors: Jiao Shoushu , Williams Phillip , Safda Naueen , Schultz Edward , Wolff Jon A.
Published: June 2017

We have previously proposed the use of primary muscle cells as a “platform,” or “vehicle” for intracerebral transgene expression. Brain grafts of minced muscle, or cultured muscle cells persisted in rat brains for at least 6 mo without any decrease in graft size, or tumor formation. Stable, but moderate levels of intracerebral transgene expression were obtained by transplanting plasmid-transfected myotubes in culture. In the present study, high and stable levels of intracerebral transgene expression were achieved by the co-transplantation of plasmid-transfected myoblasts and myotubes in culture. Approximately 5 × 10s myoblasts and myotubes were transfected with 10 μg pRSVL plasmid DNA, and 30 μg Lipofectin (BRL), respectively. They were mixed together (total cell number was 1 million), and stereotactically injected into the caudate nucleus of an adult rat brain. The activity of luciferase, the product of transgene expression, was stable for at least 4 mo, and much higher than the levels in myotube grafts, or co-grafts of myoblasts and minced muscle. Presumably, the myotubes served as a framework on which the myoblasts can form myotubes. The sections of brains transplanted with co-graft of myoblasts, and myotubes transfected with pRSVLac-Z were stained immunofluorescently for β-galactosidase activity. The muscle grafts contained β-galactosidase positive myofibers 4 mo after transplantation. Such high and stable levels of in vivo expression after postnatal gene transfer have rarely been achieved. Primary muscle cells are useful vehicle for transgene expression in brains, and potentially valuable for gene therapy of degenerative neurological disorders.
Genetically Modified Primary Astrocytes as Cellular Vehicles for Gene Therapy in the Brain
Authors: La Gamma Edmund F. , Weisinger Gary , Lenn Nicholas J. , Strecker Robert E.
Published: June 2017

Combining genetic engineering and cell transplantation has been proposed as one way to overcome the limited availability of donor tissue that may restrict the application of graft therapy to neurological diseases. Important issues in this approach concern the choice of a suitable cellular vehicle, and the method of gene insertion. In this regard, we have investigated the use of brain-derived primary astrocytes as cellular vehicles for gene therapy, because they can be transfected, divide in culture, are brain-region specific, possess a secretory mechanism, and may migrate several mm from the transplant site. To address the issue of gene insertion, we have generated stably transfected primary rat astrocytes using the nonviral calcium phosphate method to co-transfect a reporter construct (RSV-chloramphenicol acetyltransferase (CAT), or human enkephalin promoter CAT, plus a neomycin resistance plasmid (pRSVNeo). Modified astrocytes were then propagated by transfer to selective media containing G418 (300 μg/mL) for 3 wk. The presence of the reporter gene product (CAT) was demonstrated by immunocytochemistry, and by biochemical assay of CAT enzyme catalytic activity. These genetically modified astrocytes were followed for up to 3 wk after transplantation into the rat striatum. Criteria used to distinguish transplanted astrocytes included histological evidence of abundant nuclei interrupting the normal cytoarchitecture of the striatum, astrocyte morphology, and the presence of CAT enzyme activity. Our data indicates that genetically modified astrocytes are an important candidate vehicle for use in transplantation therapy in neurological diseases. We suggest that genetically modified astrocytes can also be used for studying the human enkephalin promoter, other promoters, and expressed proteins using this paradigm.
Correction of Bilirubin Conjugation in the Gunn Rat Using Hepatocytes Immobilized in Alginate Gel Beads as an Extracorporeal Bioartificial Liver
Authors: Fremond Benjamin , Malandain Christine , Guyomard Claire , Chesne Christophe , Guillouzo André , Campion Jean-Pierre
Published: June 2017

A new extracorporeal bioartificial liver using alginate-entrapped hepatocytes was developed and evaluated for its ability to correct the lack of bilirubin conjugation in the Gunn rat. Hepatocytes were harvested from Sprague-Dawley rats by the two-step collagenase perfusion method and then immobilized in Ca++-alginate beads. The ability of immobilized hepatocytes to conjugate bilirubin was investigated in vitro by comparison with hepatocyte monolayer cultures. The bioartificial liver consisted of a cylindric bioreactor containing either alginate beads with hepatocytes (test group) or alginate beads alone (control group). Gunn rats were connected to this bioreactor via an extracorporeal circulation and bile fractions were collected at hourly intervals. Both bilirubin monoconjugales and bilirubin diconjugates were measured in the bile by high pressure liquid chromatography. Hepatocyte viability in alginate beads was determined prior to and at the end of each experiment and found to be unchanged (75%). In the test group, the concentration of bilirubin conjugates increased rapidly, attaining median values of 72.26 pM and 92.59 pM for mono and diconjugated bilirubin respectively, during a 3 h period of extracorporeal circulation. In the control group, the levels of either conjugate did not exceed 0.87 μM throughout the experiments. Statistical analysis showed a significant difference between the two groups (p < 0.0023). These results suggest that the bioartificial liver used in this study represents an effective method for the temporary correction of the Gunn rat's genetic defect. Such a system might be of therapeutic interest in acute liver failure.
A 16-Channel Automated Rotometer System for Reliable Measurement of Turning Behavior in 6-Hydroxydopamine Lesioned and Transplanted Rats
Authors: Hudson John L. , Levin Dennis R. , Hoffer Barry J.
Published: June 2017

Unilateral 6-hydroxydopamine lesions of the nigrostriatal pathway in rats result in a massive dopamine (DA) denervation of the ipsilateral striatum. Such animals have proven extremely useful as a model for the study of Parkinson's disease, an idiopathic neurodegenerative disorder of humans. Extensive unilateral DA disruption leaves the rat relatively normal in motor behavior; however, the extent of the lesion can be documented by drug-induced rotational behavior. When given an injection of a dopamine agonist, such as apomorphine or d-amphetamine, a lesioned animal will manifest rotational behavior; the number of turns correlates with the degree of unilateral denervation. In order to identify, for various studies, large numbers of animals with specific levels of denervation, the necessity of an automated and reliable rotational counting system (rotometer) becomes obvious. We have developed such a device that allows up to 16 rats to be tested concurrently with one inexpensive computer. This system is more reliable than, and certainly preferable to, more tedious methods such as videotaping and subsequent manual analysis or various other mechanical systems. Plexiglass, formed into large bowls, serve as the rotometer chambers. We have designed a simple, inexpensive, and accurate counting head that can be manufactured from readily available parts and that is very sturdy and reliable. This, together with a thoracic harness, completes the rotometer assembly. The rotational data, from up to 16 separate channels, is collected by a single-chip microprocessor and sent on a serial line to an IBM-type or Macintosh host computer. There, it is graphically displayed on line and subsequently saved to disk with a novel acquisition program. Files generated are in code readable by most spreadsheet software currently available. Therefore, rotational data can be imported to a number of different spreadsheets and macros used for analysis. In summary, the multiple-channel automation for monitoring turning behavior in rats, described here, is a simple, inexpensive and effective system for accurate and rapid data acquisition and analysis.
Ultrastructural Cytochemistry of Acetylcholinesterase Enzyme Activity in Pancreatic Tissue Transplants in Rats
Authors: Adeghate Ernest , Donáth Tibor
Published: June 2017

The distribution of acetylcholinesterase (AChE) at the ultrastructural level was investigated in normal and in pancreatic fragments transplanted for 56 days into the anterior eye chamber of heterologous rats using enzyme cytochemical methods. Acetylcholinesterase reaction products were seen on the basal surface of the acinar cells in normal pancreas. Acetylcholinesterase enzyme activity was also detected on the axolemma of the surviving nerve fibres. This enzyme reaction product forms alternating thick and thin bands on the axolemma. Some of these AChE-positive nerve fibres accompany blood vessels that also survive after transplantation. AChE were seen in cytoplasm adjacent to the surviving alpha and pancreatic polypeptide cells. We conclude that the ability of some neurons and cells to produce and or store acetylcholinesterase is still retained after transplantation of pancreatic tissue into the anterior eye chamber of rats.
Transplantation of Hepatocytes: An In-Vitro and In-Vivo Study in Canines
Authors: Rivas Pedro A. , Fabrega Alfredo J. , Schwartz Daniel , Dagiantis William , Ward Michael G. , Blanchard Jacqueline , Pollak Raymond
Published: June 2017

We isolated and transplanted hepatocytes in the canine, large animal model to evaluate hepatocyte yield and purity as well as the optimal site for hepatocyte engraftment (i.e., the spleen or the portal bed). We obtained viable, pure, single hepatocyte suspensions that were readily preserved at 4°C in University of Wisconsin (UW) solution for up to 3 days. Both intrasplenic and portal vein injection were well tolerated, with minimal recipient morbidity and mortality when 1-2 × 109 hepatocytes were injected into immunosuppressed allogeneic hosts. We noted the embolization of hepatocytes into the parenchyma of the native liver within 7 days of intrasplenic transplantation that produced a mild reversible derangement of liver function and histology. These results warrant consideration prior to clinical trials of hepatocyte transplantation in man.
Preparation of Adult Pig Pancreatic Cells: Comparative Study of Methods with or without Proteolytic Enzymes
Authors: Ohgawara Hisako , Kobayashi Akira , Chong Suk-Ja , Akaike Toshihiro , Hashimoto Yoko
Published: June 2017

Methods for the preparation of adult pig pancreatic cells using enzymatic- or auto-digestion are described, and a comparison of these methods was performed. A greater number of cells staining red with dithizone with the highest viability and insulin secretory response to glucose stimulation were obtained with dispersed pancreatic cells prepared by auto-digestion of the pancreas. On the other hand, many damaged cells were observed when the pancreas was exposed to collagenase or EDTA-Dispase and only a few cells stained red with dithizone. Insulin secretion in response to glucose stimulation was observed for up to 6 h in cells prepared using the auto-digestion procedure and an approximately 2-to 3-fold increase in insulin secretion was seen in response to glucose. Cells obtained by the collagenase or EDTA-Dispase exposure did not show a secretory response to either low (5.5 mmol/l) or high (16.7 mmol/l) glucose concentrations. This report shows that it is possible to prepare pancreatic endocrine cells from the adult pig pancreas which represents an inexpensive way to obtain cells for pancreatic cell transplantation or studies of their biochemical or physiological properties.
Transplantation of Encapsulated Bovine Chromaffin Cells in the Sheep Subarachnoid Space: A Preclinical Study for the Treatment of Cancer Pain
Authors: Joseph J.M. , Goddard M.B. , Mills J. , Padrun V. , Zurn A. , Zielinski B. , Favre J. , Gardaz J.P. , Mosimann F. , Sagen J. , Christenson L. , Aebischer P.
Published: June 2017

Chromaffin cells have been shown to release a combination of pain-reducing neuroactive compounds including catecholamines and opioid peptides. The allogeneic transplantation of chromaffin cells in the subarachnoid space has been shown to alleviate pain in various rodent models and possibly in terminal cancer patients. Because of the shortage of human cadaver donor tissue, we are investigating the possibility of transplanting xenogeneic cells in polymer capsules. In this technique, cells are surrounded by a permselective synthetic membrane whose pores are suitably sized to allow diffusion of nutrients, neurotransmitters and growth factors, but restrict the diffusion of the large molecules of the immune system and prevent contact with immunocompetent cells. The encapsulation technique therefore allows transplantation of xenogeneic tissue between species as well as retrieval of transplanted cells. Previously we have reported that encapsulated bovine chromaffin cells survive and alleviate pain in various rodent models. The purpose of the present study was to assess the feasibility of implanting a human sized device in a large animal model. Adrenals from 5 calves were surgically removed; chromaffin cells were isolated from these glands using a collagenase-based digestion-filtration technique. Cells were loaded into acrylic-based tubular (5 cm long, 920 μm wide) permselective capsules attached to silicone tethers. The capsules were maintained in vitro for at least 7 days following the encapsulation procedure. Nicotine evoked release was analyzed in a defined subgroup from each batch. One capsule was then implanted using a guiding cannula system in the lumbar subarachnoid space of each sheep for 4 (n = 5) and 8 (n = 1) wk. All capsules were retrieved intact by gentle pulling on the silicone tether. Except for one capsule, the evoked catecholamine release of the retrieved capsules was in the same range as that of other capsules from the same cohort that had been maintained in vitro. All retrieved capsules were devoid of host cell reaction. Clusters of viable cells dispersed in an alginate immobilizing matrix were observed throughout all the implanted capsules. This study demonstrates the feasibility of transplanting functional encapsulated xenogeneic chromaffin cells into the cerebrospinal fluid of a large animal model using a capsule of appropriate dimensions for human implants. We believe that these results suggest the appropriateness of human clinical trials in patients suffering from refractory terminal cancer pain.
Unrelated Donor Bone Marrow Transplants in Children
Authors: Grovas Alfred , Feig Stephen A. , O'rourke Sheryl , Valentino Leonard , Wiley Fran , Hunt Lynne , Landaw Elliot M. , Gajewski James
Published: June 2017

Only a small proportion of children who might benefit from bone marrow transplant (BMT) have an HLA-identical sibling. To provide this potentially curative therapy to patients without a matched related donor, marrow transplants using less well matched related donors or unrelated donors (identified through computerized donor registries) have been performed. We report the outcome of 24 consecutive unrelated donor BMT's performed on children. Eligible diagnosis included acute leukemia (AL) (n = 15), chronic myelogenous leukemia (CML) (n = 4), myelodysplastic syndrome (MDS) (n = 3), and severe aplastic anemia (SAA) (n = 2). All donor/recipient pairs were sero-matched at 5 or 6 of the 6 HLA A, B, and DR antigens. Several different preparative regimens were used, but fractionated total body irradiation (TBI) was used in 20 patients. All recipients received graft-versus-host-disease (GVHD) prophylaxis with cyclosporine-A (CSA), four with short course methotrexate (MTX), 14 in combination with short course MTX and methylprednisolone (MPS), and five in combination with a mouse monoclonal antibody to CD5, coupled to the A-chain of ricin (Xomazyme-65). One patient received CSA and MPS alone after a T-cell depleted marrow transplant. Twenty of 23 evaluable recipients engrafted (87%). Two patients with CML never engrafted and had autologous marrow recovery, one patient with SAA died at 128 days without evidence of engraftment, and there was one early death at day + 9. Fourteen of 20 patients (70%) with stable donor-derived hematopoiesis developed significant acute GVHD ≥ grade II). Eleven of 15 engrafted patients who survived > 100 days after BMT developed chronic GVHD (73%). Thirteen patients survive, 10 disease-free; 2 yr actuarial survival and disease-free survival are 47% and 41%, respectively. Of the 19 engrafted patients with leukemia or MDS, only three have relapsed. The actuarial relapse risk at 2 yr is 24%. Unrelated donor transplants in children are associated with an increased risk of GVHD and nonengraftment compared to matched sibling transplants. Increased donor age is significantly associated with a greater risk of acute GVHD.
Improved Functional Survival of Human Islets of Langerhans in Three-Dimensional Matrix Culture
Authors: Brendel Mathias D. , Kong Shen Shen , Alejandro Rodolfo , Mintz Daniel H.
Published: June 2017

The current study evaluates functional survival of human islets maintained in tissue culture for up to 4 wk in suspension media (CMRL-1066 with supplements) and contrasts these results with immobilizing three-dimensional matrices (agarose or alginate). The absolute number and volume of islets retrieved from agarose is significantly higher after two and four wk of culture compared to conventional free-floating media. In vitro function of islets, assessed by insulin/DNA content, insulin secretion into the culture media over 24 h and glucose-theophylline stimulated insulin release in a dynamic perifusion system, was not significantly different between free-floating and matrix preserved islets. In vivo islet function was evaluated by the effectiveness for reversal of insulin-dependent diabetes mellitus by transplantation of the islets under the kidney capsule of nude mice. Although adequate insulin responses to glucose were seen after culture in conventional or matrix media, only agarose embedded islets were consistently able to induce normoglycemia in diabetic recipients after 14 days of culture. Additional transplantation experiments defined the threshold level required to reverse diabetes to be between 1,000 and 1,500 agarose preserved islets. Our data suggest improved engraftment of human islets after agarose culture. This culture method may be of benefit for the accumulation of functionally competent human islets, thus facilitating the implementation of clinical protocols that utilize freshly isolated islets from multiple donors without the need for cryopreservation.
Aberrant Function and Long-Term Survival of Mouse β Cells Exposed In Vitro to High Glucose Concentrations
Authors: Pehuet-Figoni Martine , Ballot Eric , Bach Jean-François , Chatenoud Lucienne
Published: June 2017

In vitro culture of murine Langerhans islets usually ends in islet death after 1-3 wk. Given contradictory published data, we studied the influence of glucose on the function and survival of islets from DBA/2 mice. Islets were cultured on plastic microwells, using 1, 2, or 11 g/l glucose concentrations. Using our routine technique, insulin secretion was evaluated after islet incubation for 15 min in basal medium [(bIS), 1 g/1 glucose], followed by 15 min in stimulating medium [(sIS), 3 g/l glucose, 20 mM/l arginine, 5 mM/l theophylline]. Insulin secretion of islets cultured in 1 g/l glucose remained stable and normal over a period of 2 mo [Day 7: bIS, 6.3 ± 3.1 pU/50 μl; sIS, 16.6 ± 6.8 μU/50 μl. Day 60: bIS, 6.0 ± 4.0 pU/50 μl; sIS, 21.3 ± 10.5 μU/50 μl]. Islet morphology also remained normal. Islets cultured in 2 g/l glucose showed elevated insulin response under basal and stimulating conditions during 2-3 wk, followed by a dramatic drop in insulin secretion [Day 7: bIS, 19.5 ± 5.7 μU/50 μl; sIS, 80.9 ± 10.7 μU/50 μl. Day 60: bIS, 5.4 ± 5.0 μU/50 μl; sIS, 2.7 ± 1.4 μU/50 μl]. Severe morphologic alterations appeared rapidly and islet destruction was nearly complete by 60 days. At 11 g/l glucose, functional and morphological islet alterations were accelerated [Day 7: bIS, 10.3 ± 2.7 μU/50 μl; sIS, 18.8 ± 4.9 μU/50 μl. Day 21: bIS and sIS almost undetectable]. On the basis of the results obtained in our experimental conditions, it may be concluded that the optimal glucose concentration for long-term murine islet cultures is 1 g/l. Based on the results of this study and testing an extracellular matrix, we thereafter cultured islets from DBA/2 mice at the 1 g/l glucose concentration. These islets remained functional for a period of time > 100 days. Abbreviations: bIS: insulin secretion in basal medium; sIS: insulin secretion in stimulating medium; MEM: minimal essential medium.
Repeated Intraportal Hepatocyte Transplantation in Analbuminemic Rats
Authors: Rozga Jacek , Holzman Michael , Moscioni Albert D. , Fujioka Hikaro , Morsiani Eugenio , Demetriou Achilles A.
Published: June 2017

The optimal site for implantation of isolated hepatocytes has not been established. We have developed a novel technique which allows repeated infusion of hepatocytes into the portal system via an indwelling catheter. Seven Nagase Analbuminemic rats (NAR) underwent single intra-portal infusion of 2 × 107 isolated normal albumin-producing rat hepatocytes. Another seven NAR rats underwent placement of indwelling catheters into the portal venous system via the gastroduodenal vein. Each of them received six batches of 5 × 106 normal albumin producing hepatocytes. Seven control NAR rats were infused repeatedly (intraportally) with saline only. Plasma albumin (ELISA) showed significant increase in experimental animals and was more pronounced (p < 0.05) in rats transplanted repeatedly than in those given a single dose of cells. Immunohistochemical staining of the liver sections confirmed the presence of transplanted albumin producing hepatocytes. Rats transplanted with a single large batch of isolated hepatocytes showed liver tissue damage, whereas those subjected to repeated cell infusions had normal liver histology. We have developed a novel intraportal transplantation method which allows successful engraftment of a large number of isolated hepatocytes.
Protective Effect of Glutathione (GSH) over Glutathione Monoethyl-Ester (GSH-E) on Cold Preservation of Isolated Rat Liver Cells
Authors: Rodríguez Joaquín V. , Mamprin María E. , Guibert Edgardo E. , Labadié Guillermo
Published: June 2017

Hepatocyte suspensions provide a rapid method to determine how hypothermic storage affects liver cell metabolism and viability. We investigated whether reduced Glutathione (GSH) inclusion into a modified University of Wisconsin (UW) solution, has a protective effect over Glutathione derivatives, such as Glutathione-monoethylester (GSH-E), when suspensions of hepatocytes are cold stored for several days. Isolated rat liver cells were cold preserved 96 h in UW, UW plus 3 mM GSH and UW plus 3 mM GSHE. During the cold storage, not significant changes in cell viability were observed, but the total Glutathione content was higher in systems with extracellular GSH over those with GSH-E or without. After cold storage, the liver cells were gently resuspended in Krebs-Henseleit — 1% Albumin and used for 120 min of normothermic (37°C) incubation. We evaluate the functional response of the cells measuring the exclusion of Trypan Blue (TBE). This response was clearly different in preserved cells in presence of GSH. These results indicate a protective role of extracellular Glutathione, due to an accumulation of it, rather than the derivative, for hepatic cell during the cold storage in UW solutions. And also, it is possible to extend experiments with hepatocytes from a single cell isolation over 4 or more consecutive days
Importance of pH in Resuspension Media on Viability of Hepatocytes Preserved in University of Wisconsin Solution
Authors: Mamprin María Eugenia , Rodríguez Joaquín Valentín , Guibert Edgardo Elvio
Published: June 2017

The effect of different pH of resuspension media on the viability of hepatocytes preserved (for 96 h at 4°C) in University of Wisconsin solution (UW solution) was analyzed. After this cold resuspension media storage, we evaluated the rewarming step (incubation time 120 min at 37°C) using different pH levels (6.80, 7.00, 7.20, and 7.40). Cell viability assessed by trypan blue exclusion (TBE) showed a significant difference (p < 0.05) for cells incubated at pH = 7.20. For instance, TBE expressed as percent of change was 78.1 ± 1.4 compared with cells tested at other pH (pH = 6.80, TBE = 44.2 ± 9.5; pH = 7.00, TBE = 66.5 ± 1.1 and pH = 7.40, TBE = 62.0 ± 1.4). We also evaluated the capacity of these cells both to maintain potassium content (0.509 ± 0.230 μEq. K+/106 cells) and to synthesize urea (5.36 ± 1.81 μmol Urea/106 cells). These results were compared with those obtained from freshly isolated non preserved hepatocytes (0.518 ± 0.060 μEq. K+/106 cells and 5.91 ± 0.43 μmol Urea/106 cells). The results show that viability is pH dependent and suggest that when resuspension media were used, the viability of hepatocytes was improved after 96 h of cold storage.
Collagenase Lot Selection and Purification for Adipose Tissue Digestion
Authors: Williams Stuart K. , Mckenney Sandra , Jarrell Bruce E.
Published: June 2017

Crude Clostridial collagenase (CCC) remains the most widely used enzyme for the digestion of tissues prior to cell isolation and culture. CCC contains numerous components in addition to specific collagenases and proteases. A chronic problem associated with CCC is significant lot variability which occurs with respect to the ability of different lots of CCC to digest tissue. We have evaluated numerous commercially available samples of CCC for their ability to digest human liposuction-derived SC fat. Digestion capacity was evaluated as the ability to release endothelial cells from fat as well as the ability of isolated cells to adhere to tissue culture plastic. A significant variation in digestion efficacy between lots of collagenase was observed. We subsequently purified CCC using a partial purification method with dialysis and centrifugation as well as a complete purification, using liquid chromatography, to remove all nonspecific proteases. While partially purified collagenase retained digestion capacity, pure collagenase exhibited reduced digestion capacity. Maximum digestion was achieved with pure collagenase when trypsin was added. The use of completely purified collagenase with trypsin is advantageous where all components in the enzyme digestion mixture must be known.
Microglia: The Effector Cell for Reconstitution of the Central Nervous System following Bone Marrow Transplantation for Lysosomal and Peroxisomal Storage Diseases
Authors: Krivit William , Sung Joo Ho , Shapiro Elsa G. , Lockman Lawrence A.
Published: June 2017

Treatment and potential cure of lysosomal and peroxisomal diseases, heretofore considered fatal, has become a reality during the past decade. Bone marrow transplantation, (BMT), has provided a method for replacement of the disease-causing enzyme deficiency. Cells derived from the donor marrow continue to provide enzyme indefinitely. Several scores of patients with diseases as diverse as metachromatic leukodystrophy, adrenoleukodystrophy, globoid cell leukodystrophy, Hurler syndrome (MPS I-H), Maroteaux-Lamy (MPS VI) Gaucher disease, and fucosidosis have been successfully treated following long-term engraftment. Central nervous system (CNS) manifestations are also prevented or ameliorated in animal models of these diseases following engraftment from normal donors. The microglial cell system has been considered to be the most likely vehicle for enzyme activity following bone marrow engraftment. Microglia in the mature animal or human are derived from the newly engrafted bone marrow. Graft-v-host disease activation of the microglia is also of importance. This article will summarize some of the pertinent literature relative to the role of microglia in such transplant processes.
Neural Transplantation
Authors: Lindvall Olle
Published: June 2017

Cell transplantation is now being explored as a new therapeutic strategy to restore function in the diseased human central nervous system. Neural grafts show long-term survival and function in patients with Parkinson's disease but the symptomatic relief needs to be increased. Cell transplantation seems justified in patients with Huntington's disease and, at a later stage, possibly also in demyelinating disorders. The further development in this research field will require systematic studies in animal experiments but also well-designed clinical trials in small groups of patients.
Endothelial Cell Transplantation
Authors: Williams Stuart K.
Published: June 2017

Endothelial cells line the lumenal surface of al) elements of the vascular system. These cells exhibit numerous metabolic functions necessary for the maintenance of homeostasis. The critical role of endothelium in maintaining normal blood vessel function is exemplified by the poor clinical performance of small diameter polymeric vascular grafts which fail due, in part, to the lack of a functional endothelium on the lumenal surface. Extensive research has explored the potentiality of transplanting endothelial cells onto polymeric vascular grafts to improve graft function. Several critical issues have been explored including the source of endothelial cells for transplantation, the interaction of endothelium with polymers and the healing process of endothelial cell transplanted grafts. The future of endothelial cell transplantation will also include the use of these cells as vehicles for genetic engineering.
Professional Standards in Cell and Tissue Processing
Authors: Linden Jeanne V. , Favreau Thomas J.
Published: June 2017

In the United States, standards for cell and tissue processing have been developed by a variety of professional tissue banking organizations. Several organizations, including the American Association of Tissue Banks and the Eye Bank Association of America, have accreditation programs for member institutions. Some governmental agencies, such as the New York State Department of Health and the Food and Drug Administration, have adopted strict regulations, which may subject noncompliant tissue banks to certain enforcement actions. Professional tissue banking organizations have also issued guidelines that provide recommendations for implementing efficacious policies and procedures for the acquisition, processing, storage, and distribution of tissues.
Application of Quality Assurance Practices in Processing Cells and Tissues for Transplantation
Authors: Newman-Gage Helen
Published: June 2017

Attention to issues of quality assurance from the early stages of development of experimental cell therapies provides a margin of safety for recipients. Adherence to minimum standards of practice at acquisition, processing, storage, and implantation ensures not only this baseline safety factor for patients, but also provides a baseline for comparative evaluations between different studies or different banks. This paper describes the basic components of a quality assurance program tailored to laboratories and facilities that collect, process, or distribute human cells and tissues for transplantation. These components include policy and procedure manuals, donor screening practices, processing procedures evaluation and control, training and education programs, auditing and investigation roles, responsibility for release of grafts, and recordkeeping and traceability requirements. References and resources for detailed information related to good manufacturing practices and good clinical and laboratory practices are provided. Standards, regulations, and current legislation specifically related to human cells and tissues intended for transplantation are also discussed.
Homing and Immunogenicity of Murine Stromal Cells Transfected with Xenogeneic Mhc Class II Genes
Authors: Huss Ralf , Smith Franklin O. , Myerson David H. , Deeg H. Joachim
Published: June 2017

Syngeneic (murine) and xenogeneic (canine) marrow-derived stromal cells were injected intravenously into SCID and normal mice to examine the homing pattern and persistence of these cells in vivo. By in situ hybridization, these stromal cells were detectable in the bone marrow cavity and the spleen 21 days after injection. Xenogeneic cells did not persist in normal mice but persisted in SCID mice. Conditioning of the recipients with irradiation or S-fluorouracil (5-FU) treatment did not alter these results. In addition, syngeneic murine stromal cells were transfected with the genes for canine MHC class II (DRA + DRB) and transplanted into murine recipients to investigate their homing pattern and immunogenicity. These transfected syngeneic stromal cells did also home to marrow and spleen even in normal recipients. However, these cells led to sensitization of the host towards canine antigens as shown by accelerated skin graft rejection and delayed type hypersensitivity (DTH). Thus, immunodeficient (SCID) mice allow for the homing of xenogeneic stromal cells to hemopoietic organs and for prolonged persistence. In immunocompetent (normal) mice, no xenogeneic stromal cells were identified in spleen and marrow, either because of their inability to home or more likely because of immunological rejection. In contrast, syngeneic stromal cells expressing xenogeneic MHC class II genes did home to spleen and marrow and persisted even though the recipient had become sensitized. Their survival may be due to a loss of expression of the transfected gene. Alternatively, the presentation of these xenogeneic gene products in the hemopoietic organs was such that a cytotoxic response was not induced. These results also show that stromal cells can serve as a vehicle for gene delivery, conceivably with the possibility of organ targeting.
Sources of Human Hematopoietic Stem Cells for Transplantation–A Review
Authors: Amos T.A.S. , Gordon M.Y.
Published: June 2017

Transplantation of hematopoietic stem cells provides a means of replacing a defective hematopoietic system in patients with a wide range of malignant and nonmalignant disorders that affect the blood forming tissue. The same procedure has also allowed dose-escalation of standard chemotherapy and radiotherapy in the treatment of malignant disease of nonhematological origin. Until recently, bone marrow has been the sole source of hematopoietic stem cells, but limitations of conventional bone marrow transplantation have stimulated a search for alternative sources and uses of stem cells. Fetal tissues (especially liver) are a recognized source of transplantable stem cells and offer the great advantage of reduced immunogenicity, potentially removing the problems of tissue type matching. Umbilical cord blood is also a rich source of stem cells and, although it contains alloreactive cells, it is readily available without special ethical constraints. Both fetal tissue and cord blood suffer the disadvantages of limited numbers of stem cells per donation, and there is much interest in the development of technologies for the safe and reliable expansion and/or pooling of stem and progenitor cells. The observation that small numbers of stem cells are found in the peripheral blood of adults has led to the exploitation of the blood as a further source of stem cells. The ability to mobilize these cells from the medullary compartment into the periphery by the use of chemotherapy and/or recombinant hematopoietic growth factors has enabled the collection of sufficient numbers of cells for transplantation purposes. All of these advances are increasing the options and the range of choices available to clinicians and patients in the arena of hematopoietic stem cell transplantation.
Self-Organized, Tubular Hybrid Vascular Tissue Composed of Vascular Cells and Collagen for Low-Pressure-Loaded Venous System
Authors: Hirai Jiro , Matsuda Takehisa
Published: June 2017

A tubular, hierarchically structured hybrid vascular tissue composed of vascular cells and collagen was prepared. First, a cold mixed solution of bovine aortic smooth muscle cells (SMCs) and Type I collagen was poured into a tubular glass mold composed of a mandrel and a sheath (example of dimensions: inner diameter, 1.5 mm; outer diameter, 7 mm; length, 7 cm). Upon incubation at 37°C, an SMC-incorporated collagenous gel was formed. After the sheath was removed, the resulting fragile tissue, when cultured in medium, thinned in a time-dependent manner to form an opaque, dense tissue. Higher SMC seeding density and lower initial collagen concentration induced more rapid and prominent shrinkage of the tissue. Morphologic investigation showed that over time, bipolarly elongated SMCs and collagen fiber bundles became positioned around the mandrel. Both components became circumferentially oriented. When the mandrel was removed, a tubular hybrid medial tissue was formed. A hybrid vascular tissue with a hierarchical structure was constructed by seeding endothelial cells onto the inner surface of the hybrid medial tissue. Prepared tissues tolerated luminal pressures as great as 100 mmHg and mechanical stress applied during an anastomotic procedure. This method allowed us to prepare a tubular hybrid medial tissue of predetermined size (inner diameter, wail thickness, and length) by selecting appropriate mold design, initial collagen concentration, and SMC seeding density. Such hybrid vascular tissues may provide physiological functions when implanted into the venous system.
Venous Reconstruction Using Hybrid Vascular Tissue Composed of Vascular Cells and Collagen: Tissue Regeneration Process
Authors: Hirai Jiro , Matsuda Takehisa
Published: June 2017

In this study, a tubular hybrid vascular tissue composed of vascular cells and collagen was implanted as a venous substitute, and its remodeling process was histologically investigated. First, a hybrid medial tissue was prepared by pouring a cold mixed solution of canine jugular smooth muscle cells (SMCs) and Type I collagen into a tubular glass mold and subsequent incubation at 37°C. Culture in medium for 10 days produced a dense tubular tissue. Seeding of jugular endothelial cells (ECs) onto the luminal surface of the tissue produced a hybrid vascular tissue with a hierarchical structure. These vascular tissues (inner diameter, 7 mm; length, 3 cm; wall thickness, 1 mm; n = 14) were implanted autologously in the canine posterior vena cava wrapped in Dacron mesh for up to 24 wk. Nine of 14 grafts were patent throughout implantation. In patent grafts, monolayered ECs were oriented in the direction of blood flow at 1 wk. Circumferentially oriented SMCs accumulated at the subendothelial layer and ingrown fibroblasts were sparsely distributed throughout the wall at 12 wk. Contractile phenotype of SMCs was evident at 24 wk. Collagen fibrils, which were sparsely distributed at an early period of implantation, gradually assembled to form fibrous meshes at 24 wk. Sheet-like elastic lamellae were also observed at this time. Marked wall thinning was observed at 12 and 24 wk. The resultant tissues became highly dense. The specific gravity of tissues increased with time, and reached those of natural vessels at 24 wk. Tissue remodeling progressed in a time-dependent manner and appeared to be almost complete within 6 mo of implantation.
Expression and Inducibility of Cytochrome P450 Iiia Family within Intrasplenically Transplanted Fetal Hepatocytes
Authors: Kato Kazuya , Hodgson W. John B. , Abraham Nader G. , Onodera Kazhuiko , Imai Masato , Kasai Shinichi , Mito Michio
Published: June 2017

With the development of transplantation of hepatocytes into the spleen, interest has focused on the metabolic changes associated with hepatocyte proliferation. As these changes are important for drug metabolism in hepatocytes, we examined the expression and inducibility of the cytochrome P450 IIIA family within transplanted hepatocytes. Fetal hepatocytes were harvested at 20 days of gestation from spontaneously hypertensive rats (SHRs) and transplanted into recipient adult SHR spleens. Microscopic examination of the recipient spleens 4 and 10 wk after transplantation revealed masses of hepatocytes with cordlike structures in the red pulp. Proliferating hepatocytes were detected with a bromodeoxyuridine (BrdU) immunohistochemical stain. Immunochemical studies detected cytochromes (cytos) P450 p and P450 HLp in fetal hepatocytes before transplantation without prior induction. And although these cytos were not detected by 10 wk after transplantation, they were induced with dexamethasone. These results demonstrated that fetal hepatocytes can be transplanted successfully into recipient spleens and suggested that fetal hepatocytes grow in the spleen, similar to the adult hepatocyte response.
Grafts of Fibroblasts Genetically Modified to Secrete Ngf, Bdnf, Nt-3, or Basic Fgf Elicit Differential Responses in the Adult Spinal Cord
Authors: Nakahara Yasushi , Gage Fred H. , Tuszynski Mark H.
Published: June 2017

Neuronal and axonal responses to neurotrophic factors in the developing spinal cord have been relatively well characterized, but little is known about adult spinal responses to neurotrophic factors. We genetically modified primary rat fibroblasts to produce either nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), or basic fibroblast growth factor (bFGF), then grafted these neurotrophic factor-secreting cells into the central gray matter of the spinal cord in adult rats. Spinal cord lesions were not made prior to grafting. From 2 wk to 6 mo later, sensory neurites of dorsal root origin extensively penetrated NGF-, NT-3-, and bFGF-producing grafts, whereas BDNF-secreting grafts elicited no growth responses. Putative noradrenergic neurites also penetrated NGF-secreting cell grafts. Local motor and corticospinal motor axons did not penetrate any of the neurotrophic factor-secreting grafts. These results indicate that unlesioned or minimally lesioned adult spinal cord sensory and putative noradrenergic populations retain significant neurotrophic factor responsiveness, whereas motor neurites are comparatively resistant even to those neurotrophic factors to which they exhibit survival dependence during development. Grafts of genetically modified cells can be a useful tool for characterizing neurotrophic factor responsiveness in the adult spinal cord and designing strategies to promote axonal regeneration after injury.