Effective Islet Isolation Method with Extremely High Islet Yields from Adult Pigs

Achieving good islet isolation is one of the most important factors for successful islet transplantation. Porcine pancreas is suitable for islet isolation research due to its anatomical and physiological similarities to human pancreas. In this study, we evaluated a new porcine islet isolation method designed to maximize islet yield and compared it with our previous open pan method and the standard method using a Ricordi chamber (Ricordi method). We performed 15 porcine islet isolations, five each with the new method, the open pan method, and the Ricordi method. The new method features several important improvements. Pancreata remain uncut and are kept intact during collagenase intraductal injection, a large filtration chamber to handle whole pancreata, low concentration of collagenase (Liberase™ HI) for digestion, and large plastic containers for large-scale islet purification. All isolated islets were assessed for yield, purity, viability and in vitro function. Islets isolated with this new method were transplanted under the kidney capsules of SCID mice with chemically induced diabetes for in vivo functional assessment (n = 8). With the new method, we obtained on average more than 1,000,000 islet equivalents (IE) (1,236,266 ± 213,486 IE) (mean ± SE) before purification and 800,000 IE (879,815 ± 222,729 IE) after purification from one adult pig. Islet yield per pancreas was significantly higher compared with our previous open pan method (30,666 ± 11,532 IE, p < 0.01) and the Ricordi method (317,073 ± 86,093 IE, p < 0.05). All mice, transplanted with 1000 islets from the new method, returned to normoglycemia within 4 days after transplantation. Our new method makes it possible to obtain extremely high porcine islet yield with good function. It should produce useful information for human islet isolation and transplantation, and might be applied to single donor clinical xenogeneic transplantation.

Download Full-text

The Effect of UW Solution and Its Components on the Collagenase Digestion of Human and Porcine Pancreas

Cell Transplantation ◽

10.1177/096368979500400611 ◽

1995 ◽

Vol 4 (6) ◽

pp. 615-619 ◽

Cited By ~ 27

Author(s):

Harold H. Contractor ◽

Paul R. V. Johnson ◽

David R. Chadwick ◽

Gavin S. M. Robertson ◽

Nicholas J. M. London

Keyword(s):

Cold Storage ◽

Species Difference ◽

Human Pancreas ◽

Islet Isolation ◽

Porcine Pancreas ◽

Uw Solution ◽

Porcine Islet ◽

University Of Wisconsin ◽

Collagenase Digestion ◽

Tissue Dissociation

University of Wisconsin (UW) solution is used extensively as a cold storage solution during the procurement and transport of the pancreas prior to islet isolation. However, it has been observed that UW inhibits the collagenase digestion phase of human but not porcine islet isolation, resulting in poor islet yields and islets of poor viability. The aim of this study was, therefore, to confirm this species difference and to determine which components of UW are responsible for the inhibition in the human. In the initial experiment, blocks of human and porcine pancreas (n = 7) were incubated in test tubes containing collagenase at a concentration of 4 mg/mL at 37°C dissolved in 4 mL of either Hanks' solution or UW. Every 5 min the tubes were manually shaken and the degree of tissue dissociation scored on a scale of + and +++. Our results confirm the inhibition of collagenase digestion in the human but not the pig. Using the same methodology, we then investigated the components of UW that were causing the observed inhibition in the human pancreas (n = 7). This time the collagenase was dissolved in individual or combinations of UW components. Using Hank's as a control, the results were then expressed as a median ratio. The components found to be most inhibitory were magnesium, the Na+/K+ ratio, hydroxyethyl starch (HES), and adenosine. Allopurinol in combination with either lactobionate or glutathione was markedly inhibitory (i.e., median ratio 1.8 and 1.9, respectively). The most inhibitory solution tested was a combination of the three components raffinose, glutathione, and lactobionate (median ratio 2.1). This combination was almost as inhibitory as UW itself (median ratio 2.7). These findings are essential for the development of effective cold-storage solutions for the human pancreas that do not inhibit the subsequent collagenase digestion phase of islet isolation.

Download Full-text

Purification of the Antihemophilic Factor by Gel Filtration on Agarose

Thrombosis and Haemostasis ◽

10.1055/s-0038-1651394 ◽

1969 ◽

Vol 22 (03) ◽

pp. 577-583 ◽

Cited By ~ 3

Author(s):

M.M.P Paulssen ◽

A.C.M.G.B Wouterlood ◽

H.L.M.A Scheffers

Keyword(s):

Factor Viii ◽

Plasma Proteins ◽

Large Scale ◽

Gel Filtration ◽

Large Scale Preparation ◽

Scale Preparation ◽

Antihemophilic Factor

SummaryFactor VIII can be isolated from plasma proteins, including fibrinogen by chromatography on agarose. The best results were obtained with Sepharose 6B. Large scale preparation is also possible when cryoprecipitate is separated by chromatography. In most fractions containing factor VIII a turbidity is observed which may be due to the presence of chylomicrons.The purified factor VIII was active in vivo as well as in vitro.

Download Full-text

Naphthoquinones and derivatives for chemotherapy: perspectives and limitations of their anti-trypanosomatids activities

Current Pharmaceutical Design ◽

10.2174/1381612826666201109111802 ◽

2020 ◽

Vol 26 ◽

Author(s):

Luíza Dantas-Pereira ◽

Edézio F. Cunha-Junior ◽

Valter V. Andrade-Neto ◽

John F. Bower ◽

Guilherme A. M. Jardim ◽

...

Keyword(s):

Large Scale ◽

Neglected Tropical Diseases ◽

Folk Medicine ◽

Leishmania Species ◽

Parasitic Infections ◽

Mechanistic Analysis ◽

Leishmania Spp ◽

Nanomolar Range

: Chagas disease, Sleeping sickness and Leishmaniasis, caused by trypanosomatids Trypanosoma cruzi, Trypanosoma brucei and Leishmania spp., respectively, are considered neglected tropical diseases, and they especially affect impoverished populations in the developing world. The available chemotherapies are very limited and a search for alternatives is still necessary. In folk medicine, natural naphthoquinones have been employed for the treatment of a great variety of illnesses, including parasitic infections. This review is focused on the anti-trypanosomatid activity and mechanistic analysis of naphthoquinones and derivatives. Among all the series of derivatives tested in vitro, naphthoquinone-derived 1,2,3-triazoles were very active on T. cruzi infective forms in blood bank conditions, as well as in amastigotes of Leishmania spp. naphthoquinones containing a CF3 on a phenyl amine ring inhibited T. brucei proliferation in the nanomolar range, and naphthopterocarpanquinones stood out for their activity on a range of Leishmania species. Some of these compounds showed a promising selectivity index (SI) (30 to 1900), supporting further analysis in animal models. Indeed, high toxicity to the host and inactivation by blood components are crucial obstacles to be overcome to use naphthoquinones and/or their derivatives for chemotherapy. Multidisciplinary initiatives embracing medicinal chemistry, bioinformatics, biochemistry, and molecular and cellular biology need to be encouraged to allow the optimization of these compounds. Large scale automated tests are pivotal for the efficiency of the screening step, and subsequent evaluation of both the mechanism of action in vitro and pharmacokinetics in vivo are essential for the development of a novel, specific and safe derivative, minimizing adverse effects.

Download Full-text

Lentiviral Vectors for Delivery of Gene-Editing Systems Based on CRISPR/Cas: Current State and Perspectives

Viruses ◽

10.3390/v13071288 ◽

2021 ◽

Vol 13 (7) ◽

pp. 1288

Author(s):

Wendy Dong ◽

Boris Kantor

Keyword(s):

Large Scale ◽

Lentiviral Vector ◽

Clinical Applications ◽

Scale Production ◽

Base Editing ◽

Epigenome Editing ◽

Vector Systems ◽

Dividing Cells

CRISPR/Cas technology has revolutionized the fields of the genome- and epigenome-editing by supplying unparalleled control over genomic sequences and expression. Lentiviral vector (LV) systems are one of the main delivery vehicles for the CRISPR/Cas systems due to (i) its ability to carry bulky and complex transgenes and (ii) sustain robust and long-term expression in a broad range of dividing and non-dividing cells in vitro and in vivo. It is thus reasonable that substantial effort has been allocated towards the development of the improved and optimized LV systems for effective and accurate gene-to-cell transfer of CRISPR/Cas tools. The main effort on that end has been put towards the improvement and optimization of the vector’s expression, development of integrase-deficient lentiviral vector (IDLV), aiming to minimize the risk of oncogenicity, toxicity, and pathogenicity, and enhancing manufacturing protocols for clinical applications required large-scale production. In this review, we will devote attention to (i) the basic biology of lentiviruses, and (ii) recent advances in the development of safer and more efficient CRISPR/Cas vector systems towards their use in preclinical and clinical applications. In addition, we will discuss in detail the recent progress in the repurposing of CRISPR/Cas systems related to base-editing and prime-editing applications.

Download Full-text

Pan-ERBB kinase inhibition augments CDK4/6 inhibitor efficacy in oesophageal squamous cell carcinoma

Gut ◽

10.1136/gutjnl-2020-323276 ◽

2021 ◽

pp. gutjnl-2020-323276

Author(s):

Jin Zhou ◽

Zhong Wu ◽

Zhouwei Zhang ◽

Louisa Goss ◽

James McFarland ◽

...

Keyword(s):

Squamous Cell Carcinoma ◽

Cell Carcinoma ◽

Squamous Cell ◽

Large Scale ◽

Cell Cancer ◽

Oesophageal Squamous Cell Carcinoma ◽

Squamous Cancer ◽

Striking Success

ObjectiveOesophageal squamous cell carcinoma (OSCC), like other squamous carcinomas, harbour highly recurrent cell cycle pathway alterations, especially hyperactivation of the CCND1/CDK4/6 axis, raising the potential for use of existing CDK4/6 inhibitors in these cancers. Although CDK4/6 inhibition has shown striking success when combined with endocrine therapy in oestrogen receptor positive breast cancer, CDK4/6 inhibitor palbociclib monotherapy has not revealed evidence of efficacy to date in OSCC clinical studies. Herein, we sought to elucidate the identification of key dependencies in OSCC as a foundation for the selection of targets whose blockade could be combined with CDK4/6 inhibition.DesignWe combined large-scale genomic dependency and pharmaceutical screening datasets with preclinical cell line models, to identified potential combination therapies in squamous cell cancer.ResultsWe identified sensitivity to inhibitors to the ERBB family of receptor kinases, results clearly extending beyond the previously described minority of tumours with EGFR amplification/dependence, specifically finding a subset of OSCCs with dual dependence on ERBB3 and ERBB2. Subsequently. we demonstrated marked efficacy of combined pan-ERBB and CDK4/6 inhibition in vitro and in vivo. Furthermore, we demonstrated that squamous lineage transcription factor KLF5 facilitated activation of ERBBs in OSCC.ConclusionThese results provide clear rationale for development of combined ERBB and CDK4/6 inhibition in these cancers and raises the potential for KLF5 expression as a candidate biomarker to guide the use of these agents. These data suggested that by combining existing Food and Drug Administration (FDA)-approved agents, we have the capacity to improve therapy for OSCC and other squamous cancer.

Download Full-text

A therapeutic vascular conduit to support in vivo cell-secreted therapy

npj Regenerative Medicine ◽

10.1038/s41536-021-00150-2 ◽

2021 ◽

Vol 6 (1) ◽

Author(s):

Edward X. Han ◽

Hong Qian ◽

Bo Jiang ◽

Maria Figetakis ◽

Natalia Kosyakova ◽

...

Keyword(s):

Vascular Graft ◽

Large Scale ◽

Biological Effects ◽

Therapeutic Protein ◽

Close Proximity ◽

Delivery Platform ◽

Stage Renal Disease ◽

End Stage

AbstractA significant barrier to implementation of cell-based therapies is providing adequate vascularization to provide oxygen and nutrients. Here we describe an approach for cell transplantation termed the Therapeutic Vascular Conduit (TVC), which uses an acellular vessel as a scaffold for a hydrogel sheath containing cells designed to secrete a therapeutic protein. The TVC can be directly anastomosed as a vascular graft. Modeling supports the concept that the TVC allows oxygenated blood to flow in close proximity to the transplanted cells to prevent hypoxia. As a proof-of-principle study, we used erythropoietin (EPO) as a model therapeutic protein. If implanted as an arteriovenous vascular graft, such a construct could serve a dual role as an EPO delivery platform and hemodialysis access for patients with end-stage renal disease. When implanted into nude rats, TVCs containing EPO-secreting fibroblasts were able to increase serum EPO and hemoglobin levels for up to 4 weeks. However, constitutive EPO expression resulted in macrophage infiltration and luminal obstruction of the TVC, thus limiting longer-term efficacy. Follow-up in vitro studies support the hypothesis that EPO also functions to recruit macrophages. The TVC is a promising approach to cell-based therapeutic delivery that has the potential to overcome the oxygenation barrier to large-scale cellular implantation and could thus be used for a myriad of clinical disorders. However, a complete understanding of the biological effects of the selected therapeutic is absolutely essential.

Download Full-text

Human Oligodendrocytes and Myelin In Vitro to Evaluate Developmental Neurotoxicity

International Journal of Molecular Sciences ◽

10.3390/ijms22157929 ◽

2021 ◽

Vol 22 (15) ◽

pp. 7929

Author(s):

Megan Chesnut ◽

Thomas Hartung ◽

Helena Hogberg ◽

David Pamies

Keyword(s):

Large Scale ◽

In Vitro Models ◽

Environmental Chemicals ◽

Developmental Neurotoxicity ◽

In Vivo Studies ◽

Regulatory Guidelines ◽

In Vitro Systems ◽

Human Oligodendrocytes

Neurodevelopment is uniquely sensitive to toxic insults and there are concerns that environmental chemicals are contributing to widespread subclinical developmental neurotoxicity (DNT). Increased DNT evaluation is needed due to the lack of such information for most chemicals in common use, but in vivo studies recommended in regulatory guidelines are not practical for the large-scale screening of potential DNT chemicals. It is widely acknowledged that developmental neurotoxicity is a consequence of disruptions to basic processes in neurodevelopment and that testing strategies using human cell-based in vitro systems that mimic these processes could aid in prioritizing chemicals with DNT potential. Myelination is a fundamental process in neurodevelopment that should be included in a DNT testing strategy, but there are very few in vitro models of myelination. Thus, there is a need to establish an in vitro myelination assay for DNT. Here, we summarize the routes of myelin toxicity and the known models to study this particular endpoint.

Download Full-text

In vitro multiplication, micromorphological studies and ex vitro rooting of Hybanthus enneaspermus (L.) F. Muell. – a rare medicinal plant

Acta Botanica Croatica ◽

10.1515/botcro-2017-0012 ◽

2018 ◽

Vol 77 (1) ◽

pp. 80-87 ◽

Cited By ~ 3

Author(s):

Mahipal S. Shekhawat ◽

M. Manokari

Keyword(s):

Medicinal Plant ◽

Large Scale ◽

Nodal Segments ◽

Ms Medium ◽

Ex Vitro ◽

Culture Bottle ◽

Hybanthus Enneaspermus ◽

Half Strength

AbstractHybanthus enneaspermusis a rare medicinal plant. We defined a protocol for micropropagation,ex vitrorooting of cloned shoots and their acclimatization. Surface-sterilized nodal segments were cultured on Murashige and Skoog (MS) medium with different concentrations of 6-benzylaminopurine (BAP) and kinetin (Kin). Medium supplemented with 1.5 mg L−1BAP was found optimum for shoot induction from the explants and 6.4±0.69 shoots were regenerated from each node with 97% response. Shoots were further proliferated maximally (228±10.3 shoots per culture bottle with 7.5±0.43 cm length) on MS medium augmented with 1.0 mg L−1each of BAP and Kin within 4–5 weeks. The shoots were rootedin vitroon half strength MS medium containing 2.0 mg L−1indole-3 butyric acid (IBA). The cloned shoots were pulse-treated with 300 mg L–1 of IBA and cultured on soilrite® in a greenhouse. About 96% of the IBA-pulsed shoots rootedex vitroin soilrite®, each shoot producing 12.5±0.54 roots with 5.1±0.62 cm length. Theex vitrorooted plantlets showed a better rate of survival (92%) in a field study thanin vitrorooted plantlets (86%). A comparative foliar micromorphological study ofH. enneaspermuswas conducted to understand the micromorphological changes during plant developmental processes fromin vitrotoin vivoconditions in terms of variations in stomata, vein structures and spacing, and trichomes. This is the first report onex vitrorooting inH. enneaspermusand the protocol can be exploited for conservation and large-scale propagation of this rare and medicinally important plant.

Download Full-text

Rare variants in KDR, encoding VEGF Receptor 2, are associated with tetralogy of Fallot

Genetics in Medicine ◽

10.1038/s41436-021-01212-y ◽

2021 ◽

Author(s):

Doris Škorić-Milosavljević ◽

Najim Lahrouchi ◽

Fernanda M. Bosada ◽

Gregor Dombrowsky ◽

Simon G. Williams ◽

...

Keyword(s):

Exome Sequencing ◽

Tetralogy Of Fallot ◽

Large Scale ◽

Rare Variants ◽

Growth Factor Receptor ◽

Vegf Receptor ◽

Loss Of Function ◽

Endothelial Growth Factor

Abstract Purpose Rare genetic variants in KDR, encoding the vascular endothelial growth factor receptor 2 (VEGFR2), have been reported in patients with tetralogy of Fallot (TOF). However, their role in disease causality and pathogenesis remains unclear. Methods We conducted exome sequencing in a familial case of TOF and large-scale genetic studies, including burden testing, in >1,500 patients with TOF. We studied gene-targeted mice and conducted cell-based assays to explore the role of KDR genetic variation in the etiology of TOF. Results Exome sequencing in a family with two siblings affected by TOF revealed biallelic missense variants in KDR. Studies in knock-in mice and in HEK 293T cells identified embryonic lethality for one variant when occurring in the homozygous state, and a significantly reduced VEGFR2 phosphorylation for both variants. Rare variant burden analysis conducted in a set of 1,569 patients of European descent with TOF identified a 46-fold enrichment of protein-truncating variants (PTVs) in TOF cases compared to controls (P = 7 × 10-11). Conclusion Rare KDR variants, in particular PTVs, strongly associate with TOF, likely in the setting of different inheritance patterns. Supported by genetic and in vivo and in vitro functional analysis, we propose loss-of-function of VEGFR2 as one of the mechanisms involved in the pathogenesis of TOF.

Download Full-text

Tetraspanin immunocapture phenotypes extracellular vesicles according to biofluid source but may limit identification of multiplexed cancer biomarkers

10.1101/2021.03.02.433595 ◽

2021 ◽

Author(s):

Rachel R. Mizenko ◽

Terza Brostoff ◽

Tatu Rojalin ◽

Hanna J. Koster ◽

Hila S. Swindell ◽

...

Keyword(s):

Flow Cytometry ◽

Extracellular Vesicles ◽

Fluorescence Detection ◽

Single Particle ◽

Cancer Biomarkers ◽

Surface Markers ◽

Isolation Method ◽

Standard Methods

AbstractTetraspanin expression of extracellular vesicles (EVs) is often used as a surrogate for their general detection and classification from background contaminants. This common practice typically assumes a consistent expression of tetraspanins across EV sources, thus obscuring subpopulations of variable or limited tetraspanin expression. While some recent studies indicate differential expression of tetraspanins across bulk isolated EVs, here we present analysis of single EVs isolated using various field-standard methods from a variety of in vitro and in vivo sources to identify distinct patterns in colocalization of tetraspanin expression. We report an optimized method for the use of antibodycapture single particle interferometric reflectance imaging sensing (SP-IRIS) and fluorescence detection to identify subpopulations according to tetraspanin expression and compare our findings with nanoscale flow cytometry. Using SP-IRIS and immunofluorescence, we report that tetraspanin profile is consistent from a given EV source regardless of isolation method, but that tetraspanin profiles are distinct across various sources. Tetraspanin profiles as measured by flow cytometry do not share similar trends, suggesting that limitations in subpopulation detection significantly impact apparent protein expression. We further analyzed tetraspanin expression of single EVs captured non-specifically, revealing that tetraspanin capture can bias the apparent multiplexed tetraspanin profile. Finally, we demonstrate that this bias can have significant impact on diagnostic sensitivity for tumor-associated EV surface markers. Our findings may reveal key insights into the complexities of the EV biogenesis and signaling pathways and better inform EV capture and detection platforms for diagnostic or other downstream use.

Download Full-text