Development of a Förster resonance energy transfer assay for monitoring bacterial collagenase triple-helical peptidase activity

Due to their efficiency in the hydrolysis of the collagen triple helix, Clostridium histolyticum collagenases are used for isolation of cells from various tissues, including isolation of the human pancreatic islets. However, the instability of clostridial collagenase I (Col G) results in a degraded Col G that has weak collagenolytic activity and an adverse effect on islet isolation and viability. A Förster resonance energy transfer triple-helical peptide substrate (fTHP) has been developed for selective evaluation of bacterial collagenase activity. The fTHP [sequence: Gly-mep-Flp-(Gly-Pro-Hyp)₄-Gly-Lys(Mca)-Thr-Gly-Pro-Leu-Gly-Pro-Pro-Gly-Lys(Dnp)-Ser-(Gly-Pro-Hyp)₄-NH₂] had a melting temperature (T_m) of 36.2 °C and was hydrolyzed efficiently by bacterial collagenase (k_cat/K_M = 25,000 s⁻¹ M⁻¹) but not by clostripain, trypsin, neutral protease, thermolysin, or elastase. The fTHP bacterial collagenase assay allows for rapid and specific assessment of enzyme activity toward triple helices and, thus, potential application for evaluating the efficiency of cell isolation by collagenases.     

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.     

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.     

Assay for radiolabeled type IV collagen in the presence of other proteins using a specific collagenase

An enzymatic assay is described which quantitates radiolabeled type IV basement membrane collagen in the presence of large amounts of other proteins. A partially purified neutral protease is used which cleaves type IV collagen into fragments at 37°C which are not precipitated at 1.3% (final concentration) trichloroacetic acid-tannic acid. The kinetics of type IV collagen digestion by this enzyme are not significantly altered by the presence of a 10-fold excess of type III collagen. [¹⁴C]Tryptophan-labeled control proteins prepared from fibroblast cultures are not degraded significantly by this protease in the presence of 2.5 mmN-ethylmaleimide. The proportion of type IV collagen in a mixture of labeled placenta collagenous proteins was calculated after separate digestions with the type IV collagenolytic activity and bacterial collagenase: this value compared favorably with the proportion of type IV collagen estimated by gel electrophoresis.     

Effect of the pancreatic digestion with liberase versus collagenase on the yield, function and viability of neonatal rat pancreatic islets

Many obstacles hinder the clinical application of pancreatic islet transplantation as a cure for diabetes mellitus. One of them is the suitable isolation method of sufficient number of healthy islets for transplantation. In this context, liberase enzyme was developed as a purified form of the traditional collagenase. It was the aim of this study to investigate the effect of liberase-digestion on the yield, function and viability of neonatal rat islets, and to compare the new enzyme with the collagenase. Glucose-stimulated insulin secretion was measured as indication of the function, insulin content as indication for the synthetic activity of islet cells and DNA as an indication of cell viability. The results showed no difference between islets isolated either with collagenase or liberase. Glucose stimulated similarly the insulin secretion in both. Stimulation index tended, without significance, to be higher (55%) in liberase-isolated islets compared with the collagenase islets (49%). The viability of both was similar. The insulin synthesis (content) tended also to be better in liberase-isolated islets. It could be concluded that liberase could be non-significantly preferred in the isolation of neonatal rat islets in comparison with collagenase.     

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

Type 2 diabetes is a chronic disease affecting 382 million people in 2013, and is expected to rise to 592 million by 2035 ¹. During the past 2 decades, the role of beta-cell dysfunction in type 2 diabetes has been clearly established ². Research progress has required methods for the isolation of pancreatic islets. The protocol of the islet isolation presented here shares many common steps with protocols from other groups, with some modifications to improve the yield and quality of isolated islets from both the wild type and diabetic Lepr^db (db/db) mice. A live-cell 2-photon imaging method is then presented that can be used to investigate the control of insulin secretion within islets.     

Human Pancreatic Islet Isolation: Part II: Purification and Culture of Human Islets

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of glucose monitoring and new insulin formulations, many individuals still develop devastating secondary complications. Pancreatic islet transplantation can restore near normal glucose control in diabetic patients ¹, without the risk of serious hypoglycemic episodes that are associated with intensive insulin therapy. Providing sufficient islet mass is important for successful islet transplantation. However, donor characteristics, organ procurement and preservation affect the isolation outcome ². At University of Illinois at Chicago (UIC) we developed a successful isolation protocol with an improved purification gradient ³. The program started in January 2004 and more than 300 isolations were performed up to November 2008. The pancreata were sent in cold preservation solutions (UW, University of Wisconsin or HTK, Histidine-Tryptophan Ketoglutarate) ^4-7 to the Cell Isolation Laboratory at UIC for islet isolation. Pancreatic islets were isolated using the UIC method, which is a modified version of the method originally described by Ricordi et al⁸. As described in Part I: Digestion and Collection of Pancreatic Tissue, human pancreas was trimmed, cannulated, perfused, and digested. After collection and at least 30 minutes of incubation in UW solution, the tissue was loaded in the cell separator (COBE 2991, Cobe, Lakewood, CO) for purification ³. Following purification, islet yield (expressed as islet equivalents, IEQ), tissue volume, and purity was determined according to standard methods ⁹. Isolated islets were cultured in CMRL-1066 media (Mediatech, Herndon, VA), supplemented with 1.5% human albumin, 0.1% insulin-transferrin-selenium (ITS), 1 ml of Ciprofloxacin, 5 ml o f 1M HEPES, and 14.5 ml of 7.5% Sodium Bicarbonate in T175 flasks at 37°C overnight culture before islets were transplanted or used for research.Open in a separate windowClick here to view.^{(52M, flv)}     

A Method for Murine Islet Isolation and Subcapsular Kidney Transplantation

Since the early pioneering work of Ballinger and Reckard demonstrating that transplantation of islets of Langerhans into diabetic rodents could normalize their blood glucose levels, islet transplantation has been proposed to be a potential treatment for type 1 diabetes ^1,2. More recently, advances in human islet transplantation have further strengthened this view ^1,3. However, two major limitations prevent islet transplantation from being a widespread clinical reality: (a) the requirement for large numbers of islets per patient, which severely reduces the number of potential recipients, and (b) the need for heavy immunosuppression, which significantly affects the pediatric population of patients due to their vulnerability to long-term immunosuppression. Strategies that can overcome these limitations have the potential to enhance the therapeutic utility of islet transplantation.Islet transplantation under the mouse kidney capsule is a widely accepted model to investigate various strategies to improve islet transplantation. This experiment requires the isolation of high quality islets and implantation of islets to the diabetic recipients. Both procedures require surgical steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol. We also briefly discuss different transplantation models: syngeneic, allogeneic, syngeneic autoimmune, and allogeneic autoimmune.     

The presence of collagenase in collagen preparations

The frequently observed instability of neutral salt solutions of native collagen extracted from various sources and partially purified by standard procedures has been studied by disc electrophoresis in polyacrylamide gel and by electron microscopic examination of segment long spacing crystallites. The phenomenon has revealed time and temperature dependency, pH optima near neutrality, and inhibition by sodium EDTA and serum. In addition, collagen breakdown has been found to be quantitatively related to the state of aggregation of the substrate, being more marked in reconstituted collagen gels than in collagen in solution. A typical pattern of animal collagenase degradation of native collagen into two fragments designated as TC^A and TC^B has been observed under certain conditions. It is concluded that the degradation of native collagen in neutral salt solution is due to a specific collagenase, and that this enzyme probably remains bound to collagen throughout the process of extraction and partial purification. Experiments with gelatin suggest that, in addition to collagenase, a nonspecific proteolytic activity may also be present in collagen preparations.     

Improved enzymatic isolation of fibroblasts for the creation of autologous skin substitutes

Summary The number of medical applications using autologous fibroblasts is increasing rapidly. We investigated thoroughly the procedure to isolate cells from skin using the enzymatic tissue dissociation procedure. Tissue digestion efficiency, cell viability, and yield were investigated in relation to size of tissue fragments, digestion volume to tissue ratio, digestion time, and importance of other protease activities present in Clostridium histolyticum collagenase (CHC) (neutral protease, clostripain, and trypsin). The results showed that digestion was optimal with small tissue fragments (2–3 mm³) and with volumes tissue ratios ≥2 ml/g tissue. For incubations ≤10 h, the digestion efficiency and cell isolation yields were significantly improved by increasing the collagenase, neutral protease, or clostripain activity, whereas trypsin activity had no effects. However, a too high proteolytic activity of one of the proteases present in CHC digestion solution or long exposure times interfered with cell viability and cell culture yields. The optimal range of CHC proteases activities per milliliter digestion solutions was determined for digestions ≤10 h (collagenase 2700–3900 Mandl U/ml, neutral protease 5100–10,000 caseinase U/ml, and clostripain 35–48 BAEE U/ml) and for longer digestions (>14 h) (collagenase 1350–3000 U/ml, neutral protease 2550–7700 U/ml, and clostripain 18–36 U/ml). Using these conditions, a maximum fibroblast expansion was achieved when isolated cells were seeded at 1×10⁴ cells/cm². These results did not only allow selection of optimal CHC batches able to digest dermal tissue with an high cell viability but also significantly increased the fibroblast yields, enabling us to produce autologous dermal tissue in a clinically acceptable time frame of 3 wk.     

Human Pancreatic Islet Isolation: Part I: Digestion and Collection of Pancreatic Tissue

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of glucose monitoring and new insulin formulations, many individuals still develop devastating secondary complications. Pancreatic islet transplantation can restore near normal glucose control in diabetic patients ¹, without the risk of serious hypoglycemic episodes that are associated with intensive insulin therapy. Providing sufficient islet mass is important for successful islet transplantation. However, donor characteristic, organ procurement and preservation affect the isolation outcome ². At University of Illinois at Chicago (UIC) we have developed a successful isolation protocol with an improved purification gradient ³. The program started in January 2004, and more than 300 isolations were performed up to November 2008. The pancreata were sent in cold preservation solutions (UW, University of Wisconsin or HTK, Histidine-Tryptophan Ketoglutarate) ^4-7 to the Cell Isolation Laboratory at UIC for islet isolation. Pancreatic islets were isolated using the UIC method, which is a modified version of the method originally described by Ricordi et al ⁸. Briefly, after cleaning the pancreas from the surrounding tissue, it was perfused with enzyme solution (Serva Collagenase + Neutral Protease or Sigma V enzyme). The distended pancreas was then transferred to the Ricordi digestion chamber, connected to a modified, closed circulation tubing system, and warmed up to 37°C. During the digestion, the chamber was shaken gently. Samples were taken continuously to monitor the digestion progress. Once free islets were detected under the microscope, the digestion was stopped by flushing cold (4°C) RPMI dilution solution (Mediatech, Herndon, VA) into the circulation system to dilute the enzyme. After being collected and washed in M199 media supplemented with human albumin, the tissue was sampled for pre-purification count and incubated with UW solution before purification. Purification process will be described in Part II: Purification and Culture of Human Islets.     

Hexose Metabolism in Pancreatic Islets: Glycogen Synthase and Glycogen Phosphorylase Activities

The activity of glycogen synthase and glycogen phosphorylase was measured in rat pancreatic islet homogenates. For this purpose, the sensitivity of current radioisotopic procedures for the assay of these enzymes in liver extracts was increased by about two orders of magnitude. Even so, the measurement of glycogen synthase and phosphorylase in islet homogenates was hampered by a potent amylase-like activity, resulting in the hydrolysis of preformed or newly formed ¹⁴C-labeled glycogen. Acarbose suppressed the latter phenomenon which was found attributable to both minute contamination of isolated islets by acinar cells and genuine α-amylase activity in purified islet β-cells. As measured by the more sensitive method in the presence of acarbose, the a/(a + b) ratio for glycogen synthase activity in islet homogenates was increased in islets preincubated in the presence as distinct from absence of D-glucose and decreased after preincubation with forskolin. These changes represented a mirror image of those evoked by D-glucose and forskolin in the a/(a + b) ratio for glycogen phosphorylase activity. It is concluded that glycogen synthesis and breakdown are regulated in the endocrine pancreas in a manner qualitatively comparable to that prevailing in hepatocytes, the possible participation of an amylase-like activity to glycogen metabolism in intact islet β-cells requiring further investigation.     

Isolation of Human Islets from Partially Pancreatectomized Patients

Investigations into the pathogenesis of type 2 diabetes and islets of Langerhans malfunction ¹ have been hampered by the limited availability of type 2 diabetic islets from organ donors². Here we share our protocol for isolating islets from human pancreatic tissue obtained from type 2 diabetic and non-diabetic patients who have undergone partial pancreatectomy due to different pancreatic diseases (benign or malignant pancreatic tumors, chronic pancreatitis, and common bile duct or duodenal tumors). All patients involved gave their consent to this study, which had also been approved by the local ethics committee. The surgical specimens were immediately delivered to the pathologist who selected soft and healthy appearing pancreatic tissue for islet isolation, retaining the damaged tissue for diagnostic purposes. We found that to isolate more than 1,000 islets, we had to begin with at least 2 g of pancreatic tissue. Also essential to our protocol was to visibly distend the tissue when injecting the enzyme-containing media and subsequently mince it to aid digestion by increasing the surface area.To extend the applicability of our protocol to include the occasional case in which a large amount (>15g) of human pancreatic tissue is available , we used a Ricordi chamber (50 ml) to digest the tissue. During digestion, we manually shook the Ricordi chamber³ at an intensity that varied by specimen according to its level of tissue fibrosis. A discontinous Ficoll gradient was then used to separate the islets from acinar tissue. We noted that the tissue pellet should be small enough to be homogenously resuspended in Ficoll medium with a density of 1.125 g/ml. After isolation, we cultured the islets under stress free conditions (no shaking or rotation) with 5% CO₂ at 37 °C for at least 48 h in order to facilitate their functional recovery. Widespread application of our protocol and its future improvement could enable the timely harvesting of large quantities of human islets from diabetic and clinically matched non-diabetic subjects, greatly advancing type 2 diabetes research.     

Collagenase-Released Non-Collagenous Proteins of Cortical Bone Matrix

Two distinct groups of non-collagenous components were isolated from rat cortical bone gelatin which had previously been digested with purified bacterial col-lagenase. One component was disulfide-bonded, strongly acidic, trypsin-labile glycoprotein aggregate with a molecular mass of more than 100, 000 daltons. When reduced with β-mercaptoethanol this protein disaggregated into subunits with a molecular mass of about 60, 000 daltons. The other components consisted of a group of polypeptides with a molecular mass of about 5, 000 daltons. The latter group was present in collagenase digests prepared from normal bone gelatin but was hardly detectable or absent in digests of gelatin prepared from either autolyzed, trypsinized or lathyritic bone, or from the residue of neutral salt extracted rat tail tendon.

A recently discovered group of non-collagenous proteins is tightly bound to the highly crosslinked insoluble structure of collagen fibrils of bone and dentin. Dische et al ¹ predicted the existence of these proteins in bone by analyzing the products of KOH hydrolysis of the EDTA-insoluble residues of cortical bone collagen. Comparable products were separated following digestion of bone matrix with collagenase by Herring², and oxidation of dentin matrix by alkaline sodium metaperiodate by Shuttleworth and Veis.³ Leaver et al ⁴ proposed the name collagenase-released proteins (CRP) for non-collagenous proteins obtained from the EDTA-insoluble residue of bone and dentin. There are at least two limitations in information about CRP in the above-cited reports. The first is that the collagenases were not certified to be protease-free. The second is that EDTA-insoluble residues of cortical bone were only partly digestable by bacterial collagenase. We report here isolation and partial characterization of CRP from a special preparation of bone matrix gelatin which is quantitatively digested by a protease-free bacterial collagenase-gelatinase purified by the method of Peterkofsky and Diegelmann.^{5, 6}     

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 °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.     

Optimization of condition(s) towards establishment of primary islet cell cultures from WNIN/Ob mutant rat

WNIN/Ob, a mutant rat strain, developed at the National Center for Laboratory Animal Sciences (NCLAS) facility of National Institute of Nutrition (NIN), is a new animal model to study the metabolic syndrome. These animals have 47% fat in their body and isolation of islets from these animals were compounded due to the formation of amorphous viscous and jelly like material which reduced the islet yield. However, islets isolated from WNIN adult (≥12 months) control rats gave a good islet recovery, under standard isolation procedures using collagenase digestion. In the present study we optimized culture conditions in WNIN/Ob rats to isolate islets with higher yield, and also established primary islet cell cultures from these mutant rats, retaining cellular integrity and functionality.     

Evaluation of a Collagen-Chitosan Hydrogel for Potential Use as a Pro-Angiogenic Site for Islet Transplantation

Islet transplantation to treat type 1 diabetes (T1D) has shown varied long-term success, due in part to insufficient blood supply to maintain the islets. In the current study, collagen and collagen:chitosan (10:1) hydrogels, +/- circulating angiogenic cells (CACs), were compared for their ability to produce a pro-angiogenic environment in a streptozotocin-induced mouse model of T1D. Initial characterization showed that collagen-chitosan gels were mechanically stronger than the collagen gels (0.7kPa vs. 0.4kPa elastic modulus, respectively), had more cross-links (9.2 vs. 7.4/µm²), and were degraded more slowly by collagenase. After gelation with CACs, live/dead staining showed greater CAC viability in the collagen-chitosan gels after 18h compared to collagen (79% vs. 69%). In vivo, collagen-chitosan gels, subcutaneously implanted for up to 6 weeks in a T1D mouse, showed increased levels of pro-angiogenic cytokines over time. By 6 weeks, anti-islet cytokine levels were decreased in all matrix formulations ± CACs. The 6-week implants demonstrated increased expression of VCAM-1 in collagen-chitosan implants. Despite this, infiltrating vWF⁺ and CXCR4⁺ angiogenic cell numbers were not different between the implant types, which may be due to a delayed and reduced cytokine response in a T1D versus non-diabetic setting. The mechanical, degradation and cytokine data all suggest that the collagen-chitosan gel may be a suitable candidate for use as a pro-angiogenic ectopic islet transplant site.     

A high yield method for isolating rat islets of Langerhans using differential sensitivity to freezing

Conventional methods of isolating islets of Langerhans rely upon the differential sensitivity of the pancreatic acinar tissue vs islets to enzymatic dissociation by crude collagenase, however, the yield of intact islets obtainable with this technique is quite low. Higher yields of islets can be obtained with pharmacological or surgical methods which either destroy acinar cells or cause them to release their zymogen granules. However, because of the requirement to pretreat the donor, these methods cannot be scaled-up for potential clinical use. To overcome the limitations of the conventional isolation procedures, we exploited the differential sensitivity of acinar cells and islet cells to freezing damage. Using this approach we are able to isolate greater than 2500 islets from the pancreas of a single rat. Basically, we rapidly mince pancreatic tissue, subject the tissue to a short collagenase digestion, briefly freeze the tissue at -30 degrees C in the presence of glycerol, and immediately thaw it. Subsequent enzyme treatment digests the residual acinar tissue, collagen, DNA, and proteins. Preliminary results indicate that the islets are morphologically indistinguishable from islets isolated using conventional digestion techniques.     

Protection of rat pancreatic islet function and viability by coculture with rat bone marrow-derived mesenchymal stem cells

The maintenance of viable and functional islets is critical in successful pancreatic islet transplantation from cadaveric sources. During the isolation procedure, islets are exposed to a number of insults including ischemia, oxidative stress and cytokine injury that cause a reduction in the recovered viable islet mass. A novel approach was designed in which streptozotocin (STZ)-damaged rat pancreatic islets (rPIs) were indirectly cocultured with rat bone marrow-derived mesenchymal stem cells (rBM-MSCs) to maintain survival of the cultured rPIs. The results indicated that islets cocultured with rBM-MSCs secreted an increased level of insulin after 14 days, whereas non-cocultured islets gradually deteriorated and cell death occurred. The cocultivation of rBM-MSCs with islets and STZ-damaged islets showed the expression of IL6 and transforming growth factor-β1 in the culture medium, besides the expression of the antiapoptotic genes (Mapkapk2, Tnip1 and Bcl3), implying the cytoprotective, anti-inflammatory and antiapoptotic effects of rBM-SCs through paracrine actions.