Rat Islet Isolation Research Articles

Comparative Study of The Effects of Confounding Factors on Improving Rat Pancreatic Islet Isolation Yield and Quality.

Objective Isolated pancreatic islets are valuable resources for a wide range of research, including cell replacement studies and cell-based platforms for diabetes drug discovery and disease modeling. Islet isolation is a complex and stepwise procedure aiming to obtain pure, viable, and functional islets for in vitro and in vivo studies. It should be noted that differences in rodent strains, gender, weight, and density gradients may affect the isolated islet’s properties. We evaluated the variables affecting the rat islet isolation procedure to reach the maximum islet yield and functionality, which would be critical for further studies on islet regenerative biology. Materials and Methods The present experimental study compared the yield and purity of isolated islets from non- diabetic rats of two different strains. Next, islet particle number (IPN) and islet equivalent (IEQ) were compared between males and females, and the weight range that yields the highest number of islets was investigated. Moreover, the influence of three different density gradients, namely Histopaque, Pancoll, and Lymphodex, on final isolated islets purity and yield were assessed. Finally, the viability and functionality of isolated islets were measured. Results The IEQ, IPN, and purity of isolated islets in 15 Lister hooded rats (LHRs) were significantly (P≤0.05) higher than those of the other strains. Male LHRs resulted in significantly higher IEQ compared to females (P≤0.05). Moreover, IPN and IEQ did not significantly vary among different weight groups. Also, the utilization of Histopaque and Pancoll leads to higher yield and purity. In vivo assessments of the isolated islets presented significantly reduced blood glucose percentage in the transplanted group on days 2-5 following transplantation. ConclusionBased on these results, an optimal protocol for isolating high-quality rat islets with a constant yield, purity, and function has been established as an essential platform for developing diabetes research.

Facile mechanical shaking method is an improved isolation approach for islet preparation and transplantation.

Successful islet isolation is crucial for islet transplantation and cell treatment for type 1 diabetes. Current isolation methods are able to obtain 500–1,000 islets per rat, which results in a waste of ≥50% of total islets. In the present study, a facile mechanical shaking method for improving islet yield (up to 1,500 per rat) was developed and summarized, which was demonstrated to be more effective than the existing well-established stationary method. The present results showed that isolated islets have a maximum yield of 1,326±152 when shaking for 15 min for the fully-cannulated pancreas. For both fully-cannulated and half-cannulated pancreas in the presence of rat DNAse inhibitor, the optimal shaking time was amended to 20 min with a further increased yield of 1,344±134 and 1,286±124 islets, respectively. Furthermore, the majority of the isolated islets were morphologically intact with a well-defined surface and almost no central necrotic zone, which suggested that the condition of islets obtained via the mechanical shaking method was consistent with the stationary method. Islet size distribution was also calculated and it was demonstrated that islets from the stationary method exhibited the same size distribution as the non-cannulated group, which had more larger islets than the fully-cannulated and half-cannulated groups isolated via the shaking method. In addition, the results of glucose challenge showed that the refraction index of each group was >2.5, which indicated the well-preserved function of isolated islets. Furthermore, the transplanted islets exhibited a therapeutic effect after 1 day of transplantation; however, they failed to control blood glucose levels after ~7 days of transplantation. In conclusion, these results demonstrated that the facile mechanical shaking method may markedly improve the yield of rat islet isolation, and in vitro and in vivo investigation demonstrated the well-preserved function of isolated islets in the control of blood glucose. Therefore, the facile mechanical shaking method may be an alternative improved procedure to obtain higher islet yield for islet preparation and transplantation in the treatment of type 1 diabetes.

Enzymes for Pancreatic Islet Isolation Impact Chemokine-Production and Polarization of Insulin-Producing β-Cells with Reduced Functional Survival of Immunoisolated Rat Islet-Allografts as a Consequence.

The primary aim of this study was to determine whether normal variations in enzyme-activities of collagenases applied for rat-islet isolation impact longevity of encapsulated islet grafts. Also we studied the functional and immunological properties of rat islets isolated with different enzyme preparations to determine whether this impacts these parameters. Rat-islets were isolated from the pancreas with two different collagenases with commonly accepted collagenase, neutral protease, and clostripain activities. Islets had a similar and acceptable glucose-induced insulin-release profile but a profound statistical significant difference in production of the chemokines IP-10 and Gro-α. The islets were studied with nanotomy which is an EM-based technology for unbiased study of ultrastructural features of islets such as cell-cell contacts, endocrine-cell condition, ER stress, mitochondrial conditions, and cell polarization. The islet-batch with higher chemokine-production had a lower amount of polarized insulin-producing β-cells. All islets had more intercellular spaces and less interconnected areas with tight cell-cell junctions when compared to islets in the pancreas. Islet-graft function was studied by implanting encapsulated and free islet grafts in rat recipients. Alginate-based encapsulated grafts isolated with the enzyme-lot inducing higher chemokine production and lower polarization survived for a two-fold shorter period of time. The lower survival-time of the encapsulated grafts was correlated with a higher influx of inflammatory cells at 7 days after implantation. Islets from the same two batches transplanted as free unencapsulated-graft, did not show any difference in survival or function in vivo. Lack of insight in factors contributing to the current lab-to-lab variation in longevity of encapsulated islet-grafts is considered to be a threat for clinical application. Our data suggest that seemingly minor variations in activity of enzymes applied for islet-isolation might contribute to longevity-variations of immunoisolated islet-grafts.

Collagenase H is Crucial for Isolation of Rat Pancreatic Islets

The role(s) of collagenase G (ColG) and collagenase H (ColH) during pancreatic islet isolation remains controversial, possibly due to the enzyme blends used in the previous studies. We herein examined the role of ColG and ColH using highly pure enzyme blends of recombinant collagenase of each subtype. Rat pancreases were digested using thermolysin, together with ColG, ColH, or ColG/ColH (n = 9, respectively). No tryptic-like activity was detected in any components of the enzyme blends. The efficiency of the collagenase subtypes was evaluated by islet yield and function. Immunohistochemical analysis, in vitro collagen digestion assay, and mass spectrometry were also performed to examine the target matrix components of the crucial collagenase subtype. The islet yield was highest in the ColG/ColH group (4,101 ± 460 islet equivalents). A substantial number of functional islets (2,811 ± 581 islet equivalents) was obtained in the ColH group, whereas no islets were retrieved in the ColG group. Mass spectrometry demonstrated that ColH reacts with collagen I and III. In the immunohistochemical analysis, both collagen I and III were located in exocrine tissues, although collagen III expression was more pronounced. The collagen digestion assay showed that collagen III was more effectively digested by ColH than by ColG. The present study reveals that ColH is crucial, while ColG plays only a supporting role, in rat islet isolation. In addition, collagen III appears to be one of the key targets of ColH.

The Influence of Collagen III Expression on the Efficiency of Cell Isolation With the Use of Collagenase H

ObjectiveWe previously demonstrated that collagenase H (ColH) plays a crucial role in rat islet isolation, whereas collagenase G (ColG) plays only a supporting role. We also showed that collagen III appears to be one of the key targets of ColH based on a mass spectrometry analysis. In the present study, we investigated whether our novel findings in an islet isolation model are universally applicable for other types of cell isolation, such as a hepatocyte isolation, with the use of enzyme blends of recombinant collagenases. MethodsAs the first step, the expression of one of the main matrix components, collagen III, on rat pancreatic and hepatic tissues was assessed with the use of immunohistochemical staining. ColG and ColH were expressed in recombinant E. coli carrying expression plasmids for each collagenase. Then the efficiency of the collagenase subtype on rat hepatocyte isolation was evaluated in terms of cell yield with the use of thermolysin combined with either ColG or ColH (n = 3, respectively). ResultsThe expression of collagen III on rat hepatic tissues was dramatically lower than that of rat pancreatic tissues. In the rat hepatocyte isolation, a substantial amount of hepatocytes (0.81 ± 0.11 × 106) were obtained in the ColG group, whereas almost no hepatocytes were retrieved in the ColH group, indicating that the influence of the collagenase subtypes in rat hepatocyte isolation are completely opposite to that observed in rat islet isolation. ConclusionsConsidering that the expression of collagen III on hepatic tissues was relatively low and that almost no hepatocytes were retrieved when ColH and thermolysin were used, the present study supports our novel finding that collagen III appears to be one of the key targets of ColH in hepatocyte isolation. Therefore, the semiquantification of collagen III on the target tissues not only may positively contribute to efficient islet isolation, but also may affect other types of cell isolation by optimizing the ColH amount.

Quantification of Islet Loss and Graft Functionality During Immune Rejection by 3-Tesla MRI in a Rat Model

Because metabolic markers are not suitable for early diagnosis of islet graft dysfunction, magnetic resonance imaging (MRI) has been used to study islets that were labeled pretransplantation with superparamagnetic iron oxide nanoparticles. However, the relation between graft functionality assessed by glycemia, and MRI signal remains unclear. We transplanted hyperglycemic rats intraportally with 2500 ferucarbotran-labeled syngeneic (n=10) or allogeneic (n=12) islet equivalents or normoglycemic rats with 5000 xenogeneic human islet equivalents. Images were acquired on a clinical 3-Tesla MRI scanner. When rejection occurred on days 4 and 8 in xenogeneic and allogeneic recipients, 60% (57-68) and 55% (46-73) of the initial signal remained compared to 93% (71-104) and 82% (59-90) in syngeneic controls (P=0.006 and 0.03). With a cutoff value of 84% on day 4 for the diagnosis of allogeneic rejection, sensitivity of 91% and specificity of 70% were obtained. Based on MRI signal on day 4, treatment with antilymphocytic serum from day 4 allowed graft rescue in 75% of recipients. In this model, MRI of pretransplantation superparamagnetic iron oxide nanoparticle-labeled islet grafts allows timely diagnosis of immune rejection.

Improved Islet Yield and Function by Use of a Chloride Channel Blocker During Collagenase Digestion

Protection of pancreatic islets during isolation procedures is mandatory for successful islet transplantation. Chloride channel inhibition has been reported to prevent cell death induced by various stimuli. We examined the effects of the chloride channel blocker, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid disodium salt (DIDS) and extracellular Cl(-)-free conditions on islet isolation outcomes. Experimental groups were created based on the collagenase solutions used for Wistar rat islet isolation: control group, Hanks' balanced salt solution; DIDS group, 200 μM DIDS; and Cl(-)-free group, sodium gluconate substituted for sodium chloride. We determined whether collagenase digestion induced the death of islet cells through Cl(-) influx into the cells. We then assessed islet yield and the viability of isolated islets. We observed an increase in intracellular Cl(-) concentration under collagenase digestion conditions using a Cl(-)-sensitive fluorescent dye and subsequent rupture of islet cells. Consequently, islet yields were significantly higher in the DIDS and Cl(-)-free groups than in the control group, and islet morphology of the former groups was preserved. Of streptozotocin-induced diabetic severe combined immunodeficiency mice transplanted with a marginal dose of islets, all seven mice in the DIDS group and six of the seven mice in the Cl(-)-free group became normoglycemic, compared with two of seven mice in the control group (control vs. DIDS, P=0.010; control vs. Cl(-) free, P=0.051). Our results indicate that DIDS inhibition of Cl(-) influx into islets protects islets during digestion procedures, offering a new strategy for the improvement of islet isolation outcomes.

Improved Yield and Functional Parameters of Rat Pancreas Islets Isolated under Intramuscular Anesthesia

Intraperitoneal (IP) anesthesia is commonly used for laboratory animal experiments including rat islet isolation. However, the direct effects of anesthetics on pancreatic islets have been neglected. This study compared the islet function and recovery yield from rats that were anesthetized using IP and intramuscular (IM) injection. In addition, the lag time required to lose deep pain was measured in the following anesthetics combinations. Lewis rats were anesthetized using ketamine and xylazine (K/X) or zoletil and xylazine (Z/X). A glucose challenge test was performed on each group of prepared islets. The effect of the anesthetic agents (e.g., ketamine, zoletil, xylazine alone, and the combination of K/X and Z/X) on cell lines (rat insulinoma; RIN-5F) was investigated by determining their effect on the cell viability, the amount of insulin, and insulin mRNA expression levels of RIN-5F. The time needed for deep anesthesia in IM anesthesia was significantly shortened in comparison to IP [K/X (IM: 313 ± 66 s, IP: 371 ± 84 s) and Z/X (IM: 206 ± 76 s, IP: 245 ± 92 s)]. In addition, number of isolated islet yield by IM anesthesia was significantly improved [K/X (IM: 1530 ± 242, IP: 1245 ± 149) and Z/X (IM: 1136 ± 226, IP: 511 ± 154)]. The functions of fresh islets, indicated by the stimulation index, acquired under IM anesthesia was better preserved than that of IP. The viability and the insulin secretion of RIN-5F were decreased at 24 and 48 h. Insulin gene expression levels were decreased at 24 h as well. Anesthetics may be absorbed through the pancreas surface to the islets and have a direct effect, resulting in islet exposure and deterioration during isolation. In conclusion, for rodent islet isolation, IM anesthesia is simpler and safer in comparison to IP anesthesia.

A Practical Guide to Rodent Islet Isolation and Assessment

Pancreatic islets of Langerhans secrete hormones that are vital to the regulation of blood glucose and are, therefore, a key focus of diabetes research. Purifying viable and functional islets from the pancreas for study is an intricate process. This review highlights the key elements involved with mouse and rat islet isolation, including choices of collagenase, the collagenase digestion process, purification of islets using a density gradient, and islet culture conditions. In addition, this paper reviews commonly used techniques for assessing islet viability and function, including visual assessment, fluorescent markers of cell death, glucose-stimulated insulin secretion, and intracellular calcium measurements. A detailed protocol is also included that describes a common method for rodent islet isolation that our laboratory uses to obtain viable and functional mouse islets for in vitro study of islet function, beta-cell physiology, and in vivo rodent islet transplantation. The purpose of this review is to serve as a resource and foundation for successfully procuring and purifying high-quality islets for research purposes.

An improved method for the isolation of islets from the rat pancreas.

The conventional method for rat islet isolation was improved by injection of collagenase solution (1.3 mg/ml) into the bile duct and the concomitant dissection of the inferior vena cava, static incubation of the removed distended pancreas for 15 min at 37 degrees C and filtration of the digested tissue through two different meshes of 1190 and 590 micron in pore size. This new technique led to about 2.5 times higher yield of viable islets than the conventional method.

Perfluorohexyloctane improves long-term storage of rat pancreata for subsequent islet isolation

Pancreas oxygenation by means of the hyperoxygen carrier perfluorodecalin (PFD) has been established to prevent ischemically induced damage from cold-stored pancreata. However, large-scale studies did not confirm the promising results that had been observed in smaller donor populations. This study assessed whether islet isolation from pancreata stored for prolonged periods can be improved by utilizing the new oxygen carrier perfluorohexyloctane (F6H8) characterized by lower gravity and higher lipophilicity than PFD. Subsequent to 24 h of storage in either oxygenated PFD or F6H8, the rat pancreata were assessed for the intrapancreatic partial oxygen pressure (pO(2)) and subsequently processed with current standard procedures. The intrapancreatic pO(2) was nearly identical in rat pancreata stored either in PFD or F6H8. Nevertheless, rat islet isolation outcome was significantly increased in terms of yield, integrity, in vitro function and post-transplant outcome after transplantation in diabetic nude mice when F6H8 was used as oxygen carrier. This proof-of-concept study demonstrated in rats that islet isolation performed after long-term storage of oxygenated pancreatic tissue can be significantly improved if PFD was replaced by F6H8.

The Ratio Between Collagenase Class I and Class II Influences the Efficient Islet Release From the Rat Pancreas

Previous studies indicated different roles of collagenase class I, class II and neutral protease in the enzymatic islet release from pancreatic tissue. Because no information has been available, this study was aimed to investigate the isolation efficiency of different ratios between collagenase class II and I (C-ratio) in the rat pancreas serving as model for the human pancreas without being restricted by the large variability observed in human donors. Rat pancreata were digested using a marginal neutral protease activity and 20 PZ-U of purified collagenase classes recombined to create a C-ratio of 0.5, 1.0, or 1.5. Collagenase efficiency was evaluated in terms of isolation outcome and posttransplantation function in diabetic nude mice. The highest yield of freshly isolated islets was obtained using a C-ratio of 1.0. Purity and fragmentation of freshly isolated islets were not influenced by the C-ratio. After 24-hr culture performed for quality assessment, a marginal but significant reduction of viability was observed in islets isolated by means of a C-ratio of 0.5 and 1.5. Islet in vitro and posttransplantation function revealed no negative effect mediated by different C-ratios. The present study demonstrates that the C-ratio is of significant relevance for the outcome after enzymatic rat islet isolation. The data indicate further that purified collagenase class I or class II does not damage islet tissue even if used in excess. The present study can serve as a start for subsequent experiments in the human pancreas.

Persistent Normoglycemia in the Streptozotocin-Diabetic Rat by Syngenic Transplantation of Islets Isolated From a Single Donor With Liberase

Enzymatic digestion of donor pancreas is a vital step in islet isolation process. Recently, human and large mammalian islet isolation has been improved by Liberase. However, there are no data to show the improvement of rat islet isolation with Liberase. We hypothesized that commercially available Liberase has variable activities from batch to batch and that a short culture period might improve the function of isolated islets in vivo. We therefore isolated islets with Liberase and cultured them for a short period before transplantation to reverse diabetes in a syngenic rat model. Islets of high inbred Lewis rats were isolated with Liberase, purified by discontinuous density gradients, hand-picked, and cultured at 37 degrees C for 24 hours. The same batch of Liberase was used for all experiments in this study. Freshly isolated and cultured islets were implanted intraportally into rats rendered diabetic by streptozotocin. In this study, we were not able to reverse diabetes by syngenic transplantation of freshly isolated islets from 2 donors with Liberase in the rat model. Surprisingly, for the first time, we successfully achieved normoglycemia for more than 100 days in the diabetic rats by syngenic transplantation of the cultured islets isolated with Liberase. These normoglycemic diabetic rats showed normal glucose tolerance curves. Histologic examination of the livers of the islet recipients revealed intact islets, with numerous well-granulated insulin-containing cells and only few glucagon-expressing cells. In islets of the recipients' pancreata, the remaining native islets consisted mainly of glucagon-expressing cells, with few insulin-expressing cells in the center. We conclude that Liberase isolation followed by a short culture period may be a good substitute for collagenase in rats. Islet culture before transplantation can promote the success of single-donor-single-recipient islet transplantation to reverse diabetes in the rat model.

Optimizing Conditions for Rat Pancreatic Islets Isolation

Many procedures have been described for rat pancreatic islet isolation. Several factors contribute to the pancreatic islet isolation outcome. One of the main problems in islet isolation procedure is the formation of a viscouse, gellike structure during collagenase digestion which entraps the free islets and decrease islet yield after density gradient purification. This issue has not been addressed in most techniques described for rat islet isolation. We examined effect of various factors to eliminate formation of gellike material and improve the islets yields. Islet isolation was performed on 26 adult male Wistar Albino rats weighing between 280 and 350 g. We have observed that several factors affect pancreatic islet isolation. Optimum Collagenase enzyme concentration, maintaining pH range between 7.7 and 7.9 in digestion solution, incubation temperature at 38+/-1 degrees C and addition of Calcium ion decreased the formation of gellike materials and increased islet yield. Addition of Glycerol as a gelatin solvent has also been helpful in the reduction or complete elimination of gellike material. Precise optimization of rat islet isolation procedure is useful to improve the islet yield in islet transplantation studies.

Rat islet isolation yield and function are donor strain dependent

Effective rat islet isolation is pertinent for successful islet transplantation and islet studies in vitro. To determine which rat strain yields the highest number of pure and functional islets, four commonly used rat strains were compared with regard to islet yield, islet purity and islet function. Secretory responses were assessed by stimulation with glucose, and by stimulation with glucose plus 3-isobutyl-1-methylxanthine (IBMX). We show that rat islet function and isolation yield are donor strain dependent. Albino Oxford (AO) rats donated twice as many islets than Wistar, Lewis and Sprague Dawley (SD) rats. Stimulation with glucose plus IBMX resulted in an average five-fold increase of the stimulation index of AO, Lewis, Wistar and SD rats compared to stimulation with glucose only. AO islets had improved secretory responses after a one-week culture period, but required the addition of IBMX to glucose to elicit a distinguished stimulated insulin secretion after 2 days of culture. Islets from SD rats showed inferior results with regard to purity immediately after isolation and with regard to function after short- and after long-time culture. Because Lewis islets possessed the highest secretory response to glucose (without IBMX) immediately after isolation, Lewis rats may be preferred as islet donors for immediate use. The addition of IBMX to glucose for in vitro functional testing is recommended because it elicits high insulin secretory responses of islets regardless of the rat strain. AO rats are preferred for culture experiments since the number of experimental animals is reduced two-fold compared to Lewis, Wistar and SD rats.

Effect of University of Wisconsin solution on the rat islet isolation

Effect of University of Wisconsin solution on the rat islet isolation