bluuebunny/crossref_metadata_2025 huggingface dataset:
Behavior of Arterial Wall Cells Cultured on Periodically Stretched Substrates
Authors: Kanda Keiichi , Matsuda Takehisa
Published: June 2017

Cells in vital tissues align to form the most efficient configuration for functioning. Vascular cells of arterial walls are constantly exposed to fluid shear stress and pressure-induced periodic strain component, both of which are induced by pulsatile flow. In the present study, the effect of cyclic strain on the cellular orientation response and morphological changes of bovine arterial wall cells such as endothelial cells (ECs), smooth muscle cells (SMCs) and fibroblasts (FCs) was studied. Cells seeded onto transparent elastomeric films were subjected to periodic stretch-relaxation under various amplitudes ranging from 5 to 20% and at frequencies ranging from 15 to 120 RPM for up to 24 h. Time-lapse video-recorded images of stress-loaded cells were analyzed by a computer-aided morphometric system to quantitatively evaluate the cellular orientation responses and morphological changes. The stress-loaded cells tended to align perpendicularly to the direction of stretch with time, regardless of cellular species. More pronounced orientation was attained under operating conditions with higher amplitude and frequency of stretching. The response of SMCs and FCs advanced more rapidly than that of ECs. Meanwhile, little morphological change was observed, irrespective of stress-loading or nonloading. Understanding of mechanically induced orientation response provides a fundamental basis on tissue engineering and biomechanics.
Human Fetal Tissue Research: Practice, Prospects, and Policy
Authors: Fine Alan
Published: June 2017

Tissue from human fetal cadavers has long been used for medical research, experimental therapies, and various other purposes. Research within the last two decades has led to substantial progress in many of these areas, particularly in the application of fetal tissue transplantation to the treatment of human disease. As a result, clinical trials have now been initiated at centers around the world to evaluate the use of human fetal tissue transplantation for the therapy of Parkinson's disease, insulin-dependent diabetes mellitus, and a number of blood, immunological and, metabolic disorders. Laboratory studies suggest a much wider range of disorders may in the future be treatable by transplantation of various types of human fetal tissue. A combination of characteristics renders fetal tissue uniquely valuable for such transplantation, as well as for basic research, the development of vaccines, and a range of other applications. Although substitutes for human fetal tissue are being actively sought, for many of these applications there are at present no satisfactory alternatives. Important issues remain unresolved concerning the procurement, distribution, and use of human fetal cadaver tissue as well as the effects of such use on abortion procedures and incidence. These issues can be addressed by the introduction of appropriate guidelines or legislation, and need not be an impediment to legitimate research and therapeutic use of fetal tissue.
Low-Pressure Aspiration Abortion for Obtaining Embryonic and Early Gestational Fetal Tissue for Research Purposes
Authors: Nauert G. Michael , Freeman Thomas B.
Published: June 2017

Successful transplantation of cadaver embryonic neural tissue is highly dependent on the method used to obtain the tissue. It is important that the tissue not be contaminated bacteriologically by vaginal flora during the procedure, and that it not be disrupted mechanically. A low-pressure aspiration abortion technique has been developed that allows for the safe, effective obtainment of embryonic tissue and reduces the risk of transmitting infection. Tissue was cultured in vitro in 102 cases with minimal evidence of contamination by vaginal flora. Transplants of neural tissue into over 300 rodents have resulted in no intracranial abscesses, even in the setting of immunosuppression. The suction apparatus and low-pressure aspiration minimize disruption of the embryonic tissue. Use of low pressure adds no significant additional risk to the patient. In over 300 cases, there have been no medical complications specifically attributable to the technique. Because local anesthetic is used and sonography is not routinely required, the procedure can easily be performed in an outpatient setting during routine elective abortions, with minimal slowing or disruption of the clinic's surgical schedule. In conclusion, the low-pressure aspiration abortion technique can be safely and effectively used to obtain embryonic and early gestational fetal tissue that is almost always free from bacterial, fungal, and yeast contamination and that is frequently structurally intact. It requires no significant alteration in indications for abortion, risks, methodology, timing of the abortion, or patient management.
Differential Integrin Expression Facilitates Isolation of Human Fetal Pancreatic Epithelial Cells
Authors: Levine Fred , Beattie Gillian M. , Hayek Alberto
Published: June 2017

We have studied the expression of the β1 family of integrins in fetal and adult human pancreas. Immunohistochemical staining with a monoclonal anti-β1 antibody revealed that the epithelial cells of the human fetal pancreas express high amounts of β1 integrin, while the pancreatic stromal cells express substantially lower amounts. Islets of Langerhans from human adult pancreas also expressed high amounts of β1 integrin. Taking advantage of the extremely high affinity binding between the invasin protein of Yersinia pseudotuberculosis and many β1 integrins, we have been able to isolate highly enriched populations of fetal pancreatic epithelial cells. Epithelial-enriched cell populations retain the ability to differentiate into mature endocrine cells following transplantation into nude mice.
Interferon-γ Induced Expression of MHC Antigens Facilitates Identification of Donor Cells in Chimeric Transplant Recipients
Authors: Rao Abdul S. , Demetris Anthony J. , Qian Shiguang , Murase Noriko , Li Youping , Fung John J. , Starzl Thomas E.
Published: June 2017

After whole organ transplantation, donor bone marrow-derived cells migrate out of the graft into the recipient, leading to establishment of chimerism, which is the first step towards the subsequent induction of donor-specific tolerance. In routine immunohistochemical staining, monoclonal antibodies specific for heterotopic MHC alleles are used to identify donor and recipient cells. However, it is difficult to detect these cells using this technique in long-term allograft recipients who have a persistently low donor cell population (microchimerism). Because Interferon-gamma (IFN-γ) is known to induce expression of MHC class I and class II cell surface molecules, we used this cytokine 12-48 h before sacrifice, to facilitate the identification of donor and recipient cells in the tissues of animals transplanted with either liver (B10 → C3H) or bone marrow (LEW → BN). In long-term allograft recipients, the use of IFN-γ for as briefly as 12 h prior to sacrifice, results in marked upregulation of class I and class II antigens, leading to easy identification of ubiquitously distributed low numbers of donor cells.
A New Technique for Isolating and Culturing Human Hepatocytes from Whole or Split Livers not Used for Transplantation
Authors: Dorko Kenneth , Freeswick Paul D. , Bartoli Fabio , Cicalese Luca , Bardsley Brent A. , Tzakis Andreas , Nussler Andreas K.
Published: June 2017

Large numbers of human hepatocytes were obtained from split and whole livers by using an adaptive form of the collagenase perfusion technique employed in rodent and human biopsies. In order to guarantee a homogenous distribution of the perfusate within the whole specimen, major hepatic veins were cannulated with large bore catheters. This technique allowed for the isolation of human hepatocytes on a large scale (up to 18.5 × 109 in one case) from normal and diseased liver specimens. The yield of isolated normal viable hepatocytes is inversely proportional to the donor age. In addition, it was noted that a short time between declared death and organ harvest (cross clamp time) results in higher viability of hepatocytes. In contrast, the time of cold organ preservation did not correlate with the viability or the yield of isolated hepatocytes. We conclude that the technique presented here allows isolation of large numbers of human hepatocytes from specimens unsuitable for transplantation but very valuable for biomedical research.
Hepatocellular Transplantation into the Lung in Chronic Liver Failure following Bile Duct Obstruction in the Rat
Authors: Sandbichler P. , Erhart R. , Herbst P. , Vogel W. , Herold M. , Dietze O. , Schmid Th. , Klima G. , Margreiter R.
Published: June 2017

Injection of hepatocytes or cell-free supernatant into the lung was able to prevent death from surgically induced fulminant hepatic failure in the rat in over 90% and 53% of subjects, respectively. The aim of this study was to investigate whether this technique can be applied in chronic liver failure. Chronic liver failure was induced in Lewis rats by ligation and transection of the common bile duct, which led to cirrhosis after 3-5 wk in all animals. Four groups of animals were formed: group 1 (n = 5), normal rats, serving as control; group 2 (n = 15), cirrhotic rats, no further treatment; group 3 (n = 14), hepatocyte transplantation by injection of cell suspension transcutaneously into the right lung of cirrhotic animals four wk after bile duct ligation; group 4 (n = 17), injection of 1 mL cell-free supernatant intravenously at two-day intervals, starting 4 wk after ligation. Liver function tests, prothrombin time and serum protein levels were measured weekly before and every two days after transplantation. In group 2 all animals had died 56 (49-69) days after ligation. Survival in groups 3 and 4 was similar: all rats had died from liver failure 61 (51-72) and 60 (49-76) days following bile duct ligation. Survival rates and laboratory investigations showed no significant differences between treated and untreated cirrhotic animals. These data suggest that hepatocyte transplantation into the lung as well as supernatant injection do not have any significant effect on chronic hepatic failure, at least in the rat model.
Morphology and Function of Cultured Human Fetal Pancreatic Cells Transplanted into Athymic Mice: A Longitudinal Study
Authors: Beattie Gillian M. , Butler C. , Hayek Alberto
Published: June 2017

Islet-like cell clusters generated from human fetal pancreases between 18-24 wk gestation were able to grow and mature both morphologically and functionally after transplantation under the kidney capsule of athymic nude mice. Grafted tissue, predominantly ductal in appearance was unresponsive to glucose 2 wk after grafting. The tissue assumed the trabecular column-like architecture of adult islets by 3 mo, with a concomitant 3-4-fold increase in serum human C-peptide concentration after glucose challenge. Moreover, at that time the surface area of insulin containing cells in the graft increased 20-fold over the starting material. Exocrine components, identified by their characteristic eosinophilic secretory granules were also identified at this time. Transplanted cell clusters were capable of releasing C-peptide for at least 12 mo, when the experiment was terminated. At this time, 1 yr posttransplantation, although the proportion of insulin containing cells in the graft remained unchanged from that measured at 4 mo, a reduction in the magnitude of the C-peptide response to glucose was observed; this finding coincided with a significant mononuclear infiltrate in some areas of the transplanted tissue. In summary, human fetal pancreatic cells grown in tissue culture as cell aggregates, after transplantation under the kidney capsule, remain functionally viable for virtually the life span of immunocompromised rodents. Because of their long-term viability, the use of islet-like cell clusters is a potentially useful method to expand transplantable cells.
Single Donor Transplantation of Fresh and Cryopreserved Pancreatic Fragments into the Liver Parenchyma in Syngeneic Rats
Authors: Gao R.L. , Nguyen L.T. , Guttman F.M.
Published: June 2017

In a series of experiments on syngeneic rat islet (pancreatic fragments) transplantation we demonstrate that direct hepatic transplantation is successful to alleviate streptozotocin induced diabetes with tissue from a single donor. The experimental groups were: recipients of fresh, and cryopreserved pancreatic fragments. The fresh graft was prepared by collagenase digestion. Cryopreserved fragments were further treated by a standard freeze-thaw protocol which consists of slow cooling at 0.3°C/min to −75°C followed by transfer to −196°C, in the presence of 1.4 M Me2SO, and storage at this temperature for one day or 1 wk, and then warming them back to room temperature at a rate of 35°C/min. Streptozotocin-induced diabetes in rats can be reversed by injection of isolated pancreatic fragments from a single donor directly into the liver. No significant difference was observed between the recipients receiving fresh or cryopreserved tissue for 1 day or 1 wk. It is possible that elaborate purification itself is not conducive to successful alleviation of diabetes. This would corroborate the hypothesis that trophic factors are present in impure fragments. The direct infusion of islet fragments into the liver could allow for percutaneous administration in human transplantation.
Human Fetal Cortical Tissue Fragments Survive Grafting following One Week Storage AT +4°C
Authors: Humpel Christian , Bygdeman Marc , Olson Lars , Strömberg Ingrid
Published: June 2017

Grafting of human fetal tissue fragments has been used successfully in experimental and clinical trials. The development of techniques to store human fetal tissue fragments for longer time periods would allow to establish temporary tissue banks. We dissected several human cortical tissue fragments from one fetus and tested different storage conditions (cooling, freezing, culturing). After storage, the tissue fragments were transplanted into cavities in the cortex of host rats and the volume of the surviving grafts calculated. We report that human cortical tissue fragments grafted immediately after dissection (control group) or grafted after storage for 3 h in cryopreservation medium at room temperature survived grafting and resulted in graft sizes of 102 ± 26 mm3 and 242 ± 210 mm3, respectively, however, statistically not different. When the human cortical tissue fragments were slowly frozen and stored for 1 wk and/or when the fragments were cultured for 1 week in culture medium using a roller tube technique, grafts did not survive under our conditions. However, when the human cortical tissue fragments were stored for 1 week at +4°C in cryopreservation medium, the graft size (48 ± 24 mm3) was reduced but statistically not different from the control group. We conclude that human cortical tissue fragments can be stored at +4°C for at least 1 wk without major loss of ability to survive grafting.
A Device to Measure the Oxygen Uptake Rate of Attached Cells: Importance in Bioartificial Organ Design
Authors: Foy Brent D. , Rotem Avi , Toner Mehmet , Tompkins Ronald G. , Yarmush Martin L.
Published: June 2017

Quantification of the dependence of cellular oxygen uptake rate (OUR) on oxygen partial pressure is useful for the design and testing of bioartificial devices which utilize cells. Thus far, this information has only been obtained from suspended cells and from cells attached to microcarriers. In this work, a device was developed to obtain the dependence of OUR on oxygen partial pressure for anchorage-dependent cells cultured in standard culture dishes. The device is placed and sealed on the top of the culture dish, and holds a Clark polarographic mini-electrode flush with the bottom surface of the device. It also houses a motor to spin a magnetic stir bar within the cell chamber to insure that the medium is well-mixed. Several characteristics of the device — such as oxygen leakage into the device chamber, electrode-lag time, and linearity of the electrode at low oxygen partial pressures-were quantified and their potential effect on the values of Vm(maximal OUR) and K0.5(oxygen partial pressure at which OUR is half-maximal) were evaluated. Comparison of Vmand K0.5values obtained with this device with previously published values for suspended rat hepatocytes, Bacillus cereus, and E. coli indicated that the technique provides values accurate within 30% as long as the cell under study has a K0.5greater than approximately 1.0 mmHg. For hepatocytes cultured on 0.05 mm thickness collagen gel for 1 day (n = 4) and 3 days (n = 6), Vmwas found to be 0.38 ± 0.12 and 0.25 ± 0.09 nmol O2/s/106 cells, respectively, and K0.5was found to be 5.6 ± 0.5 and 3.3 ± 0.6 mmHg, respectively. This technique should aid in predicting bioreactor conditions such as flow rate, cell density, distance of cell from flow, and gas phase oxygen partial pressure which can lead to oxygen limitations. In addition, further studies of the effect of factors such as extracellular matrix composition, metabolic substrate, and drugs on the dependence of OUR on oxygen partial pressure for many anchorage-dependent cell types can be pursued with this technique.
In Vitro Reconstruction of Hybrid Arterial Media with Molecular and Cellular Orientations
Authors: Kanda Keiichi , Matsuda Takehisa
Published: June 2017

A hybrid medial tissue composed of a type I collagen gel, into which smooth muscle cells (SMCs) derived from bovine aortic media were 3-dimensionally (3D) embedded, was constructed around an elastomeric silicone tube (outer diameter: 8 mm). Subsequently, hybrid tissues thus prepared were subjected to three modes of mechanical stimulation in the medium: one was subjected to flotation with no disturbance (isotonic control), the second was kept isometrically (static stress) and the third was subjected to continuous periodic stretch by inflation of the embedded silicone tube which simulated arterial pulsation (dynamic stress, amplitude: 5% in inner diameter; frequency: 60 RPM). After a 5-day culture period, hybrid tissues were morphologically investigated. In control gels, polygonal SMCs and extracellular collagen fiber bundles were randomly oriented. On the other hand, upon static or dynamic stress loading, bipolar spindle-shaped SMCs and dense collagen fiber bundles were aligned circumferentially around the silicone tube, which proceeded with time. The orientations of SMCs and collagen fibers were more prominent in dynamically stressed hybrid tissues than those in statically stressed ones. The pulsatile stress-loaded hybrid medial tissue mimicked the media of native muscular arteries in terms of cellular and molecular orientations.
Dynamics of Fibrovascular Tissue Ingrowth in Hydrogel Foams
Authors: Wake M. Conley , Mikos Antonios G. , Sarakinos Georgios , Vacanti Joseph P. , Langer Robert
Published: June 2017

We have investigated and quantified the degree of fibrovascular tissue ingrowth in cylindrical poly(vinyl alcohol) (PVA) foams of 12.5 mm diameter, 5 mm thickness, and 71% porosity implanted in the mesentery of rats over a period of 25 days. Fibrovascular tissue penetrated the center of PVA foams 5 days postimplantation yet the void fraction available for cell seeding was 55% and the volume average pore diameter was 190 (±39) μm. By 10 days postimplantation the void fraction had decreased to 32% and the volume average pore diameter was 121 (±20) μm. As time elapsed fibrovascular tissue continued to expand and fill the remaining pore space. At 15 days postimplantation the void space was impractical for cell seeding and continued to decrease through the remainder of the study. Our data suggest that hydrogel foams with a polydispersed pore morphology can be prevascularized with adequate space for cell seeding as the volume of tissue penetrating the foam is limited by the smaller pores in the foam structure; however, available void space for cell seeding decreases with time.
Embedded-Culture of Pancreatic β-Cells Derived from Transgenic Mouse Insulinoma as a Potential Source for Xenotransplantation using a Diffusion Chamber
Authors: Ohgawara Hisako , Miyazaki Jun-Ichi , Karibe Sachiyo , Tashiro Fumi , Akaike Toshihiro , Hashimoto Yoko
Published: June 2017

Embedded-culture of pancreatic β-cells derived from a transgenic mouse insulinoma (MIN6 cells) was studied in vitro and in vivo. The MIN6 cells were enmeshed in an agarose-PVMA-collagen matrix for long-term maintenance. The cells formed islet-like cell clusters (ICCs) in the mixed matrix. When 10 mmol/L nicotinamide was added to these cultures the cells secreted insulin in response to various concentrations of glucose, whereas the untreated control cells were unresponsive. Both control and nicotinamide-treated MIN6 cells exhibited normal β-cell function for up to 35 days in the mixed matrix, and the cells were much better preserved with nicotinamide than without it. MIN6 cells were suspended in the mixed matrix with nicotinamide and transferred into diffusion chambers to create a bio-artificial endocrine pancreas (Bio-AEP). In streptozotocine-induced diabetic rats with implanted Bio-AEP but without any immunosuppressants, a return to normoglycaemia was observed for up to 12 wk or more after transplantation. Our results indicate that nicotinamide-treated MIN6 cells embedded in a mixed matrix should be useful for the study of xenotransplantation and the development of a bioartificial endocrine pancreas.
CD34+++ Stem/Progenitor Cells Purified from Cryopreserved Normal Cord Blood can be Transduced with High Efficiency by a Retroviral Vector and Expanded Ex Vivo with Stable Integration and Expression of Fanconi Anemia Complementation C Gene
Authors: Lu Li , Ge Yue , Li Zhi-Hua , Freie Brian , Clapp D. Wade , Broxmeyer Hal E.
Published: June 2017

A future possibility for treatment of genetic diseases may be gene therapy using autologous cord blood (CB) stem/progenitor cells. This might require cryopreservation of CB stem/progenitor cells prior to purification, gene transduction, and ex vivo expansion of cells. To address this possibility, nonadherent low density T-lymphocyte depleted (NALT-) cells from fresh or cryopreserved cord blood were sorted for CD34+++ phenotype, transduced with a recombinant retroviral vector encoding Fanconi anemia complementation C (FACC) gene, and cells expanded ex vivo in suspension culture for 7 days with growth factors. The results demonstrate: 1) high recovery of viable cells after thawing; 2) high efficiency purification of CD34+++ cells from NALT- cells prior to and after cryopreservation; 3) high degree of expansion of nucleated cells and immature progenitors from CD34+++ cells before and after cryopreservation; 4) efficient transduction with stable integration and expression of newly introduced genes in cryopreserved and then sorted stem/progenitor cells, as detected prior to and after ex vivo expansion; and 5) high efficiency transduction of single isolated CD34+++ cells obtained from cryopreserved NALT- CB. This information should be of value for future studies evaluating the use of cryopreserved cord blood for gene transfer/gene therapy.
Survival and Function of Islets during Culture
Authors: Clayton Heather A. , London Nick J.M.
Published: June 2017

Cell and tissue culture techniques have improved considerably since the first attempts to maintain explants of animal tissue in vitro. The two major developments that have allowed these improvements are the ability to produce continuous cell lines, thus allowing reproducible results to be obtained, and the definition of media for different cell types, thereby reducing the need for supplements of serum and other extraneous extracts. The requirements of islets in culture have been more difficult to define, largely because islets do not proliferate in culture and proliferation rate cannot therefore be used to measure the suitability of the medium. Further difficulties arise because islets are highly metabolically active “mini-organelles.” Although many studies have been undertaken to try and optimize media for the culture islets of Langerhans, the media most commonly used are commercially available media developed for other cell types. There remains ample scope for further refinement of the composition of islet culture media, with the possibility of different media for islets from different species.
Noradrenergic and Cholinergic Reinnervation of Islet Grafts in Diabetic Rats
Authors: Houwing Harmina , Van Asperen Roelie M. , Van Der Zee Eddy A. , Van Suylichem Paul T.R. , Oestreicher A. Beate , Steffens Anton B. , Strubbe Jan H.
Published: June 2017

Grafted islets become denervated due to the islet transplantation procedure. The aim of the present study was 1) to examine whether islet grafts in the liver, the spleen, and under the kidney capsule in rats become reinnervated following the transplantation and experimental procedures used in our laboratory, 2) whether there is any difference in reinnervation at these different sites, and 3) how these results relate to previous physiological experiments. Isogeneic isolated islets were transplanted into diabetic Albino Oxford rats, resulting in normoglycaemia. After at least 5 wk, graft-receiving organs were removed and several antibodies were employed to detect insulin, neuron-specific proteins, and cholinergic and noradrenergic nerve fibers. Islets in all three receiving organs contained viable insulin-positive B-cells. Neuron-specific enolase (NSE) as well as the growth-associated protein B-50 was observed at all sites. The cholinergic marker choline acetyltransferase (ChAT) was localized in islets grafts at all sites, but with the lowest density in the spleen. Staining for the noradrenergic markers tyrosine hydroxylase (TH) and dopamine-β-hydroxylase (DBH) was observed in islet grafts at all sites with the lowest density in grafts under the kidney capsule. All these neurochemical substances were most frequently observed in fibers associated with blood vessels, which may be the route along which nerves grow into the graft. It can be concluded that 1) islet grafts in the liver, in the spleen and under the kidney capsule become reinnervated; 2) the innervation pattern of the islet grafts differs only slightly from that in the control pancreatic islets; and 3) in combination with our previously physiological data, we can conclude that these nerve fibers are, at least partly, functionally active.
Protection of Mouse Islet Isografts from Nonspecific Inflammatory Damage by Recipient Treatment with Nicotinamide and 15-Deoxyspergualin
Authors: Kenmochi Takashi , Miyamoto Masaaki , Mullen Yoko
Published: June 2017

The major cause of primary nonfunction of transplanted islets is nonspecific inflammation associated with the transplantation procedures. Using mouse islet isografts, we attempted to prevent graft loss mediated by nonspecific inflammation using recipient treatment with nicotinamide (NA) and 15-deoxyspergualin (DSG). Newborn BALB/c islets, ranging in numbers between 1200 and 1500, were transplanted into syngeneic adult mice made diabetic by intravenous injection of 200 mg/kg streptozotocin. Recipient mice were divided into the following four groups, based on the treatment protocol of NA and DSG: intraperitoneal injection (IP) of normal saline (Group 1), IP injection of 2500 mg/kg NA (Group 2), IP injection of 5 mg/kg DSG (Group 3), and IP injection of NA + DSG (Group 4). Treatment started Day -1 and continued until Day 9 (Day 0 is day of transplantation). Blood and urine glucose, body weight, and intravenous glucose tolerance tests (IV-GTT) were examined after transplantation. Reversal of diabetes, as indicated by normoglycemia and negative urine glucose, was higher in Groups 2 (75%), 3 (50%), and 4 (85.7%), compared to Group 1 (11.1%). Especially in Group 4, the endocrine function and morphology of grafted islets were well preserved as shown by K values of IV-GTT and histological studies. These results suggest the importance of islet protection from irreversible damage by nonspecific inflammation at earlier stages of implantation, and the effectiveness of a short course of treatment with NA and DSG.
Indefinite Survival of Rat Islet Allografts following Infusion of Donor Bone Marrow without Cytoablation
Authors: Ricordi Camillo , Murase Norico , Rastellini Cristiana , Behboo Roubik , Demetris Anthony J. , Starzl Thomas E.
Published: June 2017

We have tested the effect of donor bone marrow cell (DBMC) infusion on the survival of pancreatic islet allografts in the rat, without the use of cytoablative recipient conditioning. Lewis and diabetic Brown Norway rats were used as donors and recipients, respectively. Donor islets were placed beneath the left renal capsule. Infusion of DBMC and temporary immunosuppression followed by delayed islet transplantation resulted in indefinite survival of all islet grafts (MST >180 days). Control animals demonstrated recurrent hyperglycemia (islet allografts rejection). Donor bone marrow derived cells were detected in the spleen and cervical lymph nodes of BN recipients of LEW bone marrow but not in the recipients of islet transplants alone. Second set full thickness skin grafts were performed in normal BN and in recipients of a previously successful ITX. Donor specific skin grafts were accepted in the animals that had received DBMC 40 days before the islet allograft, while animals receiving DBMC at the time of the islet allograft rejected the donor specific skin graft similarly to the controls. However, these animals did not reject a second set donor-specific islet transplant. The results indicate that radiation conditioning of the recipients was not necessary to induce microchimerism and graft acceptance in this rodent model of islet allotransplantation.
Transplantation of Human Fetal Tissue from Spontaneous Abortions to a Rodent Model of Parkinson's Disease
Authors: Kondoh Takeshi , Pundt Lisa L. , Blount Jeffrey P. , Conrad John A. , Low Walter C.
Published: June 2017

The use of human fetal tissue from elective abortions for cell transplantation therapies has been the subject of considerable controversy. Because of concerns regarding the use of tissue from elective abortions, tissue from spontaneous abortions has been suggested as an alternate donor source. In the present study we have evaluated human fetal tissue from spontaneous abortions to assess its viability, growth potential, and functional expression. Viable cells (Grade I) from a donor (7 wk postconception) were transplanted as a suspension into the striatum of rats with unilateral 6-OHDA lesions of the nigrostriatal pathway. A second group of animals received solid grafts of tissue from a Grade I donor 14 wk postconception. Tissue from Grade II and III specimens were not sufficiently viable for transplantation. Locomotor responses were monitored over a period of 15 wk and revealed an amelioration of rotational asymmetry by animals that received tissue from the 7 wk donor. Animals receiving tissue from the 14 wk donor showed no functional improvement. We found numerous graft-derived tyrosine hydroxylase (TH) immunopositive neurons contained within the transplantation site, and a rich plexus of TH-immunopositive fibers extending into the striatum of animals receiving tissue from the 7 wk donor. Animals receiving tissue from the 14 wk donor exhibited tissue necrosis at the transplant site and were devoid of TH-immunopositive neurons. These results suggest that human fetal ventral mesencephalic cells from spontaneous abortions can survive and develop after transplantation, and rectify locomotor deficits associated with experimental parkinsonism if the donor tissue is of the appropriate gestational age at the time of implantation. Our study further suggests, however, that the availability of tissue from spontaneous abortions of sufficient viability is quite limited and may thus restrict its potential use in cell transplantation therapies for Parkinson's disease.
Isolation and Culture of Porcine Hepatocytes for Artificial Liver Support
Authors: Naik Sharda , Trenkler Donna , Santangini Henry , Pan Jing , Jauregui Hugo O.
Published: June 2017

The primary requirement of cells in a liver support system is the preservation of the in vivo metabolic functions that prevent or decrease the progress of hepatic encephalopathy (HE) by providing interim support to liver failure patients. While rodent hepatocytes offer a model for liver assist device (LAD) research, their limited number per animal prohibits direct scale up to human devices. Healthy human liver cells are seldom available in adequate numbers to support clinical LAD use; consequently, a large animal source of liver cells is needed. The study presented here explored the potential of porcine hepatocytes to proliferate and maintain metabolic function in vitro. Porcine hepatocytes were isolated from ~12 kg swine by a modification of Seglen's method. Hepatocytes cultured up to 10 days were shown to metabolize ammonia and maintain both Phase I and II detoxification functions. In addition, the cultures showed proliferative activity both as an increase in total protein content and by thymidine incorporation. Immunocytochemical staining identified cell proliferation through Day 4 to be primarily hepatocytes while Days 6 and 10 showed nonparenchymal cells to be increasing. The detoxification functions measured showed peak activity on Day 4 and gradually declined through Day 10. The ability of porcine hepatocytes to proliferate and maintain a diversity of hepatic functions in culture strongly suggests their potential for use as the biological component of artificial LADs.
Effects of Intraventricular Encapsulated Hngf-Secreting Fibroblasts in Aged Rats
Authors: Lindner Mark D. , Kearns Cristin E. , Winn Shelley R. , Frydel Beata , Emerich Dwaine F.
Published: June 2017

Exogenous NGF administered into the central nervous system (CNS) has been reported to improve cognitive function in aged rats. However, concerns have been expressed about the risks involved with supplying NGF to the CNS. In this study, baby hamster kidney cells (BHK) genetically modified to secrete human NGF (hNGF) were encapsulated in semipermeable membranes and implanted intraventricularly. ChAT/LNGFR-positive basal forebrain neurons were shown to atrophy and degenerate with age, especially in cognitively impaired rats. The encapsulated BHK-NGF cells produced less than 10% of doses previously reported to be effective, but this was sufficient to increase the size of ChAT/LNGFR-positive basal forebrain neurons in the aged and learning-impaired rats to the size of the neurons in young healthy rats. The hNGF from these encapsulated cells also improved performance in a repeated-acquisition version of the Morris water maze spatial learning task in learning-impaired 20.6- and 26.7- mo-old rats. Furthermore, there was no evidence that these doses of hNGF impaired Morris water maze performance in the youngest 3.3-5.4 mo rats, and analyses of mortality rates, body weights, somatosensory thresholds, potential hyperalgesia, and activity levels, suggested that these levels of exogenous hNGF are not toxic or harmful to aged rats. These results suggest that CNS-implanted semipermeable membranes, containing genetically modified xenogeneic cells continuously producing these levels of hNGF, attenuate age-related cognitive deficits in nonimmunosuppressed aged rats, and that both the surgical implantation procedure and long-term exposure to low doses of hNGF appear safe in aged rats.
Bovine Chromaffin Cells for CNS Transplantation do not Elicit Xenogeneic T Cell Proliferative Responses in Vitro
Authors: Czech Kimberly A. , Pollak Raymond , Pappas George D. , Sagen Jacqueline
Published: June 2017

Adrenal chromaffin cells have been utilized for several neural grafting applications, but limitations in allogeneic donor availability and dangers inherent in auto-grafting limit the widespread use of this approach clinically. While xenogeneic donors offer promise as a source for cell transplantation in the central nervous system (CNS), immunologic responses to cellular components of the adrenal medulla have not been well characterized. To further study the host T cell xenogeneic response to chromaffin and passenger cells of the adrenal medulla, an in vitro lymphocyte proliferation assay was used. Lymphocyte proliferation was determined by mixing rat lymphocytes with potential stimulator cell subpopulations of the bovine adrenal medulla: isolated chromaffin cells, isolated endothelial cells, or passenger nonchromaffin cells, which include a mixture of fibroblasts, smooth muscle cells, and endothelial cells. As a positive control, bovine aortic endothelial cells were also used.3[H]-thymidine incorporation, corresponding to lymphocyte proliferation, was measured. Results indicated that isolated bovine chromaffin cells produce only a mild, statistically insignificant stimulation of rat lymphocytes. In contrast, there was a significant response to passenger nonchromaffin cells of the adrenal medulla, especially endothelial cells. The inclusion of low levels of cyclosporin A in the cultures completely eliminated the mild proliferative response to isolated bovine chromaffin cells, while near toxic levels were necessary to abrogate the response to endothelial cells. Immunocytochemical analysis revealed that routine chromaffin cell isolation procedures result in the inclusion of a small percentage of endothelial cells, which may be responsible for the slight lymphocyte stimulation. The results of this study indicate that isolated chromaffin cells possess low immunogenicity, and suggest that passenger cells in the adrenal medulla, particularly endothelial cells, may be primarily responsible for progressive rejection in CNS grafts. Thus, removal of passenger nonchromaffin cells from xenogeneic donor tissues prior to transplantation may produce a more tolerated graft in rodent models of neural transplantation.
Development of an Intact Blood-Brain Barrier in Brain Tissue Transplants is Dependent on the Site of Transplantation
Authors: Granholm Ann-Charlotte E. , Curtis Maria , Diamond David M. , Branch Berrilyn J. , Heman Karen L. , Rose Gregory M.
Published: June 2017

Transplantation of fetal septal forebrain tissue was performed to the anterior chamber of the eye, or intracranially to the rostral hippocampal formation in rats, to evaluate the impact of transplantation site on the development of an intact blood–brain barrier (BBB). The tissue was studied at 1, 2, 3, and 4 wk following transplantation by means of intravenous injection of Trypan blue, which is a vital stain not normally penetrating the BBB, as well as with an antibody specifically directed against the rat BBB, SMI71. In the intraocular septal transplants, there was a significant leakage of Trypan blue 1 wk postgrafting, associated with a few laminin-immunoreactive blood vessels that did not contain any SMI71-immunoreactivity. However, at 2 wk postgrafting, the intraocular grats exhibited an extensive plexus of thin-walled blood vessels expressing SMI71 immunoreactivity and no Trypan blue leakage. Thus, it appeared that a BBB had developed to some degree by 2 wk postgrafting in oculo. In the intracranial grafts, on the other hand, Trypan blue leakage could be seen as long as 3 wk postgrafting, and a dense plexus of blood vessels with SMI71 immunoreactivity was first seen at 4 wk postgrafting. Thus, the development of Trypan blue impermeability was delayed with 1 to 2 wk in the intracranial versus the intraocular grafts. Control experiments using psychological stress in adult rats as a means to transiently disrupt the BBB revealed that an increase in Trypan blue leakage correlated well with the disappearance of SMI71 immunoreactivity. Taken together, these studies demonstrate that the site of transplantation can influence the development of an intact BBB in neural tissue grafts.
Use of Mammalian Liver Cells for Artificial Liver Support
Authors: Jauregui Hugo O. , Chowdhury Namita Roy , Chowdhury Jayanta Roy
Published: June 2017

Advances in orthotopic liver transplantation have improved the survival rate of both acute and chronic liver failure patients to nearly 70%. However, the success of this treatment modality has created an international organ shortage. Many patients die while awaiting transplantation in part due to the minimal capacity to store viable transplantable livers beyond 24 h. Additionally, for many areas of the world, routine use of whole liver transplantation to treat liver disease is impractical due to the demands on both financial and technical resources. Potentially, these issues may be alleviated, at least in part, by the use of liver cell transplantation or cellular-based liver assist devices. The well-documented regenerative capacity of the liver may obviate the need for whole organ transplantation in some instances of acute failure, if the patient may be provided temporary metabolic support. Although other patients ultimately may require transplantation, a longer period of time to find a suitable organ for transplantation may be gained by that supportive therapy. The field of liver cell transplantation may offer solutions to patients with inherited metabolic deficiencies or chronic liver disease. The potential to treat an hepatic disorder by using only a fraction of the whole liver would increase the number of whole organs available for orthotopic liver transplantation. Research in the fields of hepatocyte based intra- and extra-corporeal liver support is providing evidence that these therapeutic modalities may ultimately become routine in the treatment of severe liver disease. A historic overview of that technology along with its current status is discussed.
Fabrication of Pliable Biodegradable Polymer foams to Engineer Soft Tissues
Authors: Wake M. Conley , Gupta Puneet K. , Mikos Antonios G.
Published: June 2017

We have fabricated pliable, porous, biodegradable scaffolds with poly(lactic-co-glycolic acid) (PLGA) and poly(ethylene glycol) (PEG) blends using a solvent-casting and particulate-leaching technique. Our study investigated the effects of four different processing parameters on pliability and pore morphology of the biodegradable scaffolds. The parameters investigated were the PLGA copolymer ratio, the PLGA/PEG blend ratio, the initial salt weight fraction, and the salt particle size. A wide range of shear moduli (0.59 to 9.55 MPa), porosities (0.798 to 0.942), and median pore diameters (71 to 154 μm) was able to be achieved by varying the combination of these parameters. Our study indicates that initial salt weight fraction and PLGA/PEG blend ratio have the most significant effects on the physico-mechanical properties of the scaffolds. Enhanced pliability of the three dimensional foams made with blends of PLGA and PEG is evidenced by the ability to roll them into a tube without macroscopic damage to the scaffold. Pliable polymer substrates hold great promise for regeneration of soft tissues such as skin, or those requiring a tubular conformation such as intestine or vascular grafts.
A Method for Estimating Number and Mass of Islets Transplanted within a Membrane Device
Authors: Suzuki Kazuhisa , Bonner-Weir Susan , Hollister Jennifer , Weir Gordon C.
Published: June 2017

Immunobarrier devices may prevent the immune destruction of pancreatic islets transplanted into diabetic recipients, but there are concerns about the survival of islets within such devices. In this manuscript we described a method for estimating islet mass and number within a membrane device. Five hundred syngeneic mouse islets were placed in a membrane device, which was then transplanted into the epididymal space of streptozotocin diabetic mouse. After 14 days the device was removed from the recipient, fixed, and embedded in paraffin. Sections were made and then stained with hematoxylin. From a total of 58 sections, 12 sample sections were selected for analysis by computerized planimetry to determine area of the device chamber and the islet area for each of these sections. By making certain assumptions, it was possible to estimate the total volume of the device chamber, the number of islets contained within the chamber, and the mass of islet tissue. Two weeks after implantation of the chamber, into which approximately 500 microencapsulated islets were loaded, the blood glucose level of the recipient fell to 73 mg/dL. The volume of the device chamber, the total volume of islets in the device, and the total islet number within the device were 1.78 μL, 0.57 μL, and 277 islets, respectively. The detailed methodology, assumptions, and calculations for this approach are described in this manuscript. This new method makes it possible to determine islet mass within a membrane device by analyzing a relatively small number of selected sections. This approach should make it possible to carry out comprehensive studies on the fate of transplanted islets contained in such immunobarrier membrane devices.
High Yield of Rodent Islets with Intraductal Collagenase and Stationary Digestion—A Comparison with Standard Technique
Authors: Shapiro A.M. James , Hao Ergeng , Rajotte Ray V. , Kneteman Norman M.
Published: June 2017

Intraductal distention of the pancreas with collagenase followed by stationary warm incubation improves the recovery of islets of Langerhans in the rat, but controlled studies are needed for valid comparison with standard isolation methods. We have modified Gotoh's technique of stationary digestion for high-yield isolation in the rat (Stationary). The method is subjected herein to rigorous blinded comparison with the standard chopped tissue (Chopped) technique, based on Lacy et al., as performed in our laboratory for over 10 yr. Islet recovery was determined by a single observer ‘blinded’ to the method of isolation used, and only intact islets of diameter ≥ 100 μm were included. Stationary gave 719 ± 114 islets per pancreas (mean ± SD, n = 21 isolations) vs. 487.5 ± 69 for Chopped (n = 36 isolations), a 47.5% increment in yield (p < 0.0001). In vitro islet perifusion showed no statistical difference in stimulation index (SI) or stimulated area under the curve (SAUC) between the two methods, but Stationary showed a trend towards improved phase II insulin release. In vivo function was assessed by isogeneic transplantation of 2,000 islets beneath the renal capsule of streptozotocin diabetic recipients (65 mg/kg Sigma); Stationary recipients (n = 7) became normoglycemic (≤ 8 mmol/L) by 3.3 ± 4.8 days vs. 1.6 ± 1.5 days for Chopped recipients (p = 0.4 ns, mean ± SEM). IVGTT performed at 1 mo posttransplant gave K-values for Stationary of 2.64 ± 0.8 vs. 2.62 ± 0.8 for Chopped (mean ± SD, p = 0.9 ns, n = 6, unpaired t-test), which were not distinguishable from normal control rats (2.59 ± 0.8) (p = 0.9 ns, n = 10). Graft function remained stable until graft bearing nephrectomy induced hyperglycemia uniformly within 1 day. Graft histology showed a healthy well-preserved structure on light microscopy, with well-granulated beta cells on EM. Economic costs of rat, collagenase, and Ficoll were 26% ($50.82) lower per recipient for Stationary. We conclude that modified stationary digestion significantly improves islet recovery with excellent in vitro and in vivo function, and is cost effective.