Nonenzymatic Cryogenic Isolation of Therapeutic Cells: Novel Approach for Enzyme-Free Isolation of Pancreatic Islets Using In Situ Cryopreservation of Islets and Concurrent Selective Freeze Destruction of Acinar Tissue

Cell-based therapies, which all involve processes for procurement and reimplantation of living cells, currently rely upon expensive, inconsistent, and even toxic enzyme digestion processes. A prime example is the preparation of isolated pancreatic islets for the treatment of type 1 diabetes by transplantation. To avoid the inherent pitfalls of these enzymatic methods, we have conceptualized an alternative approach based on the hypothesis that cryobiological techniques can be used for differential freeze destruction of the pancreas (Px) to release islets that are selectively cryopreserved in situ. Pancreata were procured from juvenile pigs using approved procedures. The concept of cryoisolation is based on differential processing of the pancreas in five stages: 1) infiltrating islets in situ preferentially with a cryoprotectant (CPA) cocktail via antegrade perfusion of the major arteries; 2) retrograde ductal infusion of water to distend the acinar; 3) freezing the entire Px solid to lt; −160°C for storage in liquid nitrogen; 4) mechanically crushing and pulverizing the frozen Px into small fragments; 5) thawing the frozen fragments, filtering, and washing to remove the CPA. Finally, the filtered effluent (cryoisolate) was stained with dithizone for identification of intact islets and with Syto 13/PI for fluorescence viability testing and glucose-stimulated insulin release assessment. As predicted, the cryoisolate contained small fragments of residual tissue comprising an amorphous mass of acinar tissue with largely intact and viable (>90%) embedded islets. Islets were typically larger (range 50–500 μm diameter) than their counterparts isolated from juvenile pigs using conventional enzyme digestion techniques. Functionally, the islets from replicate cryoisolates responded to a glucose challenge with a mean stimulation index = 3.3 ± 0.7. An enzyme-free method of islet isolation relying on in situ cryopreservation of islets with simultaneous freeze destruction of acinar tissue is feasible and proposed as a new and novel method that avoids the problems associated with conventional collagenase digestion methods.

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Abstract 63: ACE2 Deficiency Reduces Insulin Content of Isolated Pancreatic Islets and Plasma Insulin Concentrations Post-Glucose Challenge in Obese C57BL/6 Mice

Hypertension ◽

10.1161/hyp.62.suppl_1.a63 ◽

2013 ◽

Vol 62 (suppl_1) ◽

Author(s):

Robin C Shoemaker ◽

Lisa A Cassis

Keyword(s):

Gene Expression ◽

Insulin Secretion ◽

Pancreatic Islets ◽

Plasma Insulin ◽

Insulin Content ◽

Isolated Pancreatic Islets ◽

Diet Induced Obesity ◽

Glucose Challenge ◽

Ace2 Gene ◽

Deficient Mice

Objective: Diet-induced obesity promotes type 2 diabetes (T2D). Drugs that inhibit the renin-angiotensin system (RAS) have been demonstrated in clinical trials to decrease the onset of T2D. Angiotensin converting enzyme 2 (ACE2) negatively regulates the RAS by catabolizing angiotensin II (AngII). Preliminary data indicate that ACE2 deficient mice display impairments in glucose homeostasis at 8 weeks of age. We tested the hypothesis that ACE2 deficiency promotes the development of glucose intolerance and β-cell dysfunction in mice with diet-induced obesity. Methods and Results: Male Ace2 +/y or -/y mice were fed a low fat (LF, 10% kcal as fat) or high fat (HF, 60% kcal as fat) diet for 5 or 17 weeks. After 5 weeks, plasma insulin concentrations (0, 30 min) following a glucose challenge were significantly greater in HF versus ( vs) LF-fed mice. However, glucose-stimulated increases in plasma insulin concentrations were decreased in HF-fed ACE2 deficient mice compared to controls (2.96 ± 0.18 vs 4.44 ± 0.40 ng/ul, respectively; P<0.01). Surprisingly, isolated pancreatic islets from HF-fed mice of either genotype released similar concentrations of insulin in response to glucose. However, mRNA abundance of insulin was significantly reduced in islets from HF-fed Ace2 -/y compared to +/y mice (1.76 ± 0.17 vs 2.54 ± 0.18 insulin/18S ratio; P<0.05). After 17 weeks, the plasma insulin response to glucose was further reduced in the HF-fed ACE2 deficient mice compared to controls (8.07 ± 0.98 vs 13.90 ± 1.10 ng/ul; P<0.01). Further, LF-fed ACE2 deficient mice also displayed reductions in plasma glucose-stimulated insulin concentrations (1.92 ± 0.98 vs 3.09 ± 0.98 ng/ul; P<0.01). Islets from HF-fed wild type mice displayed reduced ACE2 gene expression compared to LF (0.069 ± 0.009 vs 0.169 ± 0.01, ACE2/18S ratio; P<0.001) and AngII totally suppressed islet glucose-stimulated insulin secretion compared to vehicle (-0.16 ± 0.18 vs 0.9 ± 0.26, fold change over basal; P<0.05). Conclusions: These results demonstrate that ACE2 deficiency promotes the development of T2D by regulating islet insulin content. Moreover, diet-induced obesity reduces islet ACE2 gene expression with augmented AngII-induced impairment of insulin secretion.

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91. Cryo-isolation: A novel new method for enzyme-free isolation of pancreatic islets involving in situ cryopreservation of islets and selective destruction of acinar tissue

Cryobiology ◽

10.1016/j.cryobiol.2011.09.094 ◽

2011 ◽

Vol 63 (3) ◽

pp. 331 ◽

Cited By ~ 2

Author(s):

Michael J. Taylor ◽

Simona C. Baicu

Keyword(s):

Pancreatic Islets ◽

New Method ◽

Selective Destruction ◽

Acinar Tissue ◽

Isolation Of Pancreatic Islets

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Cryo-isolation: A Novel Method for Enzyme-Free Isolation of Pancreatic Islets Involving In Situ Cryopreservation of Islets and Selective Destruction of Acinar Tissue

Transplantation Proceedings ◽

10.1016/j.transproceed.2011.10.001 ◽

2011 ◽

Vol 43 (9) ◽

pp. 3181-3183 ◽

Cited By ~ 5

Author(s):

M.J. Taylor ◽

S. Baicu

Keyword(s):

Pancreatic Islets ◽

Selective Destruction ◽

Novel Method ◽

Acinar Tissue ◽

Isolation Of Pancreatic Islets

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Equine glucagon-like peptide-1 receptor physiology

PeerJ ◽

10.7717/peerj.4316 ◽

2018 ◽

Vol 6 ◽

pp. e4316 ◽

Cited By ~ 5

Author(s):

Murad H. Kheder ◽

Simon R. Bailey ◽

Kevin J. Dudley ◽

Martin N. Sillence ◽

Melody A. de Laat

Keyword(s):

Insulin Secretion ◽

Pancreatic Islets ◽

Insulin Response ◽

Synthetic Analogue ◽

Isolated Pancreatic Islets ◽

Novel Approach ◽

Insulin Dysregulation ◽

Glucagon Like Peptide ◽

Glucagon Like Peptide 1

Background Equine metabolic syndrome (EMS) is associated with insulin dysregulation, which often manifests as post-prandial hyperinsulinemia. Circulating concentrations of the incretin hormone, glucagon-like peptide-1 (GLP-1) correlate with an increased insulin response to carbohydrate intake in animals with EMS. However, little is known about the equine GLP-1 receptor (eGLP-1R), or whether GLP-1 concentrations can be manipulated. The objectives were to determine (1) the tissue localisation of the eGLP-1R, (2) the GLP-1 secretory capacity of equine intestine in response to glucose and (3) whether GLP-1 stimulated insulin secretion from isolated pancreatic islets can be attenuated. Methods Archived and abattoir-sourced tissues from healthy horses were used. Reverse transcriptase PCR was used to determine the tissue distribution of the eGLP-1R gene, with immunohistochemical confirmation of its pancreatic location. The GLP-1 secretion from intestinal explants in response to 4 and 12 mM glucose was quantified in vitro. Pancreatic islets were freshly isolated to assess the insulin secretory response to GLP-1 agonism and antagonism in vitro, using concentration-response experiments. Results The eGLP-1R gene is widely distributed in horses (pancreas, heart, liver, kidney, duodenum, digital lamellae, tongue and gluteal skeletal muscle). Within the pancreas the eGLP-1R was immunolocalised to the pancreatic islets. Insulin secretion from pancreatic islets was concentration-dependent with human GLP-1, but not the synthetic analogue exendin-4. The GLP-1R antagonist exendin 9-39 (1 nM) reduced (P = 0.08) insulin secretion by 27%. Discussion The distribution of the eGLP-1R across a range of tissues indicates that it may have functions beyond insulin release. The ability to reduce insulin secretion, and therefore hyperinsulinemia, through eGLP-1R antagonism is a promising and novel approach to managing equine insulin dysregulation.

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In Situ LSPR Sensing of Secreted Insulin in Organ-on-Chip

Biosensors ◽

10.3390/bios11050138 ◽

2021 ◽

Vol 11 (5) ◽

pp. 138

Author(s):

María A. Ortega ◽

Júlia Rodríguez-Comas ◽

Ozlem Yavas ◽

Ferran Velasco-Mallorquí ◽

Jordina Balaguer-Trias ◽

...

Keyword(s):

Pancreatic Islets ◽

Metabolic Diseases ◽

Disease Modeling ◽

Localized Surface Plasmon ◽

Animal Testing ◽

Novel Approach ◽

On Chip ◽

External Stimuli

Organ-on-a-chip (OOC) devices offer new approaches for metabolic disease modeling and drug discovery by providing biologically relevant models of tissues and organs in vitro with a high degree of control over experimental variables for high-content screening applications. Yet, to fully exploit the potential of these platforms, there is a need to interface them with integrated non-labeled sensing modules, capable of monitoring, in situ, their biochemical response to external stimuli, such as stress or drugs. In order to meet this need, we aim here to develop an integrated technology based on coupling a localized surface plasmon resonance (LSPR) sensing module to an OOC device to monitor the insulin in situ secretion in pancreatic islets, a key physiological event that is usually perturbed in metabolic diseases such as type 2 diabetes (T2D). As a proof of concept, we developed a biomimetic islet-on-a-chip (IOC) device composed of mouse pancreatic islets hosted in a cellulose-based scaffold as a novel approach. The IOC was interfaced with a state-of-the-art on-chip LSPR sensing platform to monitor the in situ insulin secretion. The developed platform offers a powerful tool to enable the in situ response study of microtissues to external stimuli for applications such as a drug-screening platform for human models, bypassing animal testing.

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