Magnus Ståhle

Human Islet Separation Utilizing a Closed Automated Purification System

A central step within the human islet isolation process is the separation of islets from contaminating exocrine tissue utilizing linear, continuous density gradients manufactured by means of manually controlled standard gradient makers (SGM). The present study was performed to develop a closed, automated purification system (APS) that customizes density gradient profiles aiming to standardize and optimize human islet purification. Digested human pancreata were pooled, split evenly, and incubated in UW solution according to our standard protocol (n = 11). Continuous density gradient centrifugation was performed in parallel in two refrigerated COBE 2991 cell separators loaded with light (1.076 g/ml) and heavy (1.097 g/ml) Ficoll utilizing either an SGM or two computer-controlled pumps connected to Ficoll-containing bags. Quality control included islet equivalent (IE) yield, purity, in vitro function, and islet cytokine expression. Gradient profiles demonstrated that the APS readily customizes linear and nonlinear gradients. In comparison to the SGM, the APS recovered a higher percentage of the expected volume of continuous gradients (90.0 ± 1.1% vs. 98.2 ± 2.0%, p < 0.05). Islet yield (120,468 ± 15,970 vs. 114,570 ± 15,313 IE, NS) and purity (51.7 ± 4.8% vs. 54.4 ± 4.9%, NS) were nearly identical utilizing the SGM or APS. Decreased MCP-1, IL-6, and IL-8 expression indicated that APS-purified islets were possibly exposed to less proinflammatory stress. Compared to standard procedures, similar success and gentle continuous density gradient separation of human islets is feasible utilizing the APS. The APS facilitates the standardization of this complex procedure according to cGMP standards.

Pathogen inactivation of human serum facilitates its clinical use for islet cell culture and subsequent transplantation.

Serum is regarded as an essential supplement to promote survival and growth of cells during culture. However, the potential risk of transmitting diseases disqualifies the use of serum for clinical cell therapy in most countries. Hence, most clinical cell therapy programs have replaced human serum with human serum albumin, which can result in inferior quality of released cell products. Photochemical treatment of different blood products utilizing Intercept? technology has been shown to inactivate a broad variety of pathogens of RNA and DNA origin. The present study assesses the feasibility of using pathogen-inactivated, blood group-compatible serum for use in human pancreatic islet culture. Isolated human islets were cultured at 37°C for 3–4 days in CMRL 1066 supplemented with 10% of either pathogen-inactivated or nontreated human serum. Islet quality assessment included glucose-stimulated insulin release (perifusion), ADP/ATP ratio, cytokine expression, and posttransplant function in diabetic nude mice. No differences were found between islets cultured in pathogen-inactivated or control serum regarding stimulated insulin release, intracellular insulin content, and ADP/ATP ratio. Whether media was supplemented with treated or nontreated serum, islet expression of IL-6, IL-8, MCP-1, or tissue factor was not affected. The final diabetes-reversal rate of mice receiving islets cultured in pathogen-inactivated or nontreated serum was 78% and 87%, respectively (NS). As reported here, pathogen-inactivated human serum does not affect viability or functional integrity of cultured human islets. The implementation of this technology for RNA- and DNA-based pathogen inactivation should enable reintroduction of human serum for clinical cell therapy.

Shaping the tumor stroma and sparking immune activation by CD40 and 4-1BB signaling induced by an armed oncolytic virus.

Purpose: Pancreatic cancer is a severe indication with short expected survival despite surgery and/or combination chemotherapeutics. Checkpoint blockade antibodies are approved for several cancer indications, but pancreatic cancer has remained refractory. However, there are clinical data suggesting that stimulation of the CD40 pathway may be of interest for these patients. Oncolytic viruses armed with immunostimulatory genes represent an interesting approach. Herein, we present LOAd703, a designed adenovirus armed with trimerized CD40L and 4-1BBL that activates the CD40 and 4-1BB pathways, respectively. As many cells in the tumor stroma, including stellate cells and the infiltrating immune cells, express CD40 and some 4-1BB, we hypothesize that LOAd703 activates immunity and simultaneously modulates the biology of the tumor stroma. Experimental Design: Tumor, stellate, endothelial, and immune cells were infected by LOAd703 and investigated by flow cytometry, proteomics, and functional analyses. Results: LOAd703-infected pancreatic cell lines were killed by oncolysis, and the virus was more effective than standard-of-care gemcitabine. In in vivo xenograft models, LOAd703 efficiently reduced established tumors and could be combined with gemcitabine for additional effect. Infected stellate and tumor cells reduced factors that promote tumor growth (Spp-1, Gal-3, HGF, TGFβ and collagen type I), while chemokines were increased. Molecules involved in lymphocyte migration were upregulated on infected endothelial cells. Dendritic cells were robustly stimulated by LOAd703 to produce costimulators, cytokines and chemokines, and such DCs potently expanded both antigen-specific T cells and NK cells. Conclusions: LOAd703 is a potent immune activator that modulates the stroma to support antitumor responses. Clin Cancer Res; 23(19); 5846–57. ©2017 AACR.

Clostripain, the Missing Link in the Enzyme Blend for Efficient Human Islet Isolation

Background Effective digestive enzymes are crucial for successful islet isolation. Supplemental proteases are essential as they synergize with collagenase for effective pancreas digestion. The presence of tryptic-like activity has been implicated in efficient enzyme blends and the present study aimed to evaluate if addition of clostripain, an enzyme with tryptic-like activity, could improve efficacy of the islet isolation procedure. Methods Clostripain was added to the enzyme blend just before pancreas perfusion. Islets were isolated per standard method and numerous isolation parameters, islet quality control, and the number of isolations fulfilling standard transplantation criteria were evaluated. Two control organs per clostripain organ were chosen by blindly matching against body mass index, cold ischemia time, hemoglobin A1c, donor sex, and donor age. Results There were no differences in pancreas weight, dissection time, digestion time, harvest time, percent digested pancreas, or total pellet volume before islet purification between control or clostripain pancreases. Glucose-stimulated insulin release results were similar between groups. Total isolation islet equivalents, purified tissue volume and islet equivalents/g pancreas as well as fulfillment of transplantation criteria favored clostripain processed pancreases. Conclusions The addition of clostripain to the enzyme blend soundly improved islet yields and transplantation rates. It gently aided pancreas digestion and maintained proper islet functionality. The addition of clostripain to the enzyme blend has now been implemented into standard isolation protocols at the isolation centers in Uppsala and in Oslo.

Photochemical pathogen inactivation of human serum enables its large-scale application in clinical cell transplantation

Stringent protocols for cGMP, issued by regulatorybodies in Asia, Europe and North America, regulateproduction of cells and tissues for clinical therapy. Dueto the risk of transferring infections, human serum, thepreferred supplement throughout the process of cell cul-ture and transplantation, cannot be used, resulting ininferior quality of released cell products. Photochemicaltreatment (INTERCEPT Cerus Europe BV, Amersfoort,Netherlands), with the psoralen compound, amotosalenHCl and long-wavelength ultraviolet light) has beenshown to inactivate any pathogen of RNA and DNAorigin [1–4]. INTERCEPT treatment of human serumcould allow its use in processing cells and tissues forclinical application.No long-term exposure in vitro of cells to residual amo-tosalen has been performed. However, a carcinogenicitystudy in p53 knockout mice exposed to doses 1000-foldthe clinical dose for 26 weeks, showed no evidence ofcarcogenicity [5].The use of PI-HS compared with C-HS was examined onhuman melanoma cell lines as well as in the production ofclinical grade islets of Langerhans (for the treatment ofType I Diabetes), mesenchymal stem cells (for the MSCs:treatment of GvHD grade III or IV) and T cells (for immunetherapy against malignant melanoma).There was no difference in rate of proliferation during a3-day culture in medium supplemented with either PI-HSor C-HS (10%) for five human melanoma cell-lines(397mel, 526mel, SK23mel, EB81mel, CP64mel).Similarly, there was no difference between islets culturedfor 4 days inCMRL-1066 supplemented with PI-HS orC-HSintermsofinsulinstimulationindexfromadynamicperifu-sion system where the average of the high values dividedwith the average of the low values (14AE7±4AE15, 10AE0±4AE57), the ADP⁄ATP-ratio (0AE010 ± 0AE013, )0AE0004 ±0AE013) and the capacity to cure STZ-diabetic mice (78%,87%). Likewise, no difference was found in intracellularinsulin content (2AE6±1AE14, 2AE3±0AE85 ng⁄ml), expressionof IL-6 (0AE0067 ± 0AE016, 0AE0075 ± 0AE018 pmol⁄lgDNA),IL-8 (0AE264 ± 0AE150, 0AE352 ± 0AE169 pmol⁄lgDNA), MCP-1(0AE164 ± 0AE086, 0AE166 ± 0AE080 pmol⁄lgDNA) or TissueFactor(0AE034 ± 0AE007,0AE087 ± 0AE057 pmol⁄lgDNA).Also, expansion of MSC and the capacity for immuno-suppression in MLC and PHA stimulated lymphocyte cul-tures (71 ± 13, 59 ± 9%) did not differ between MSCgenerated in CMRL-1066 supplemented with PI-HS orC-HS.Finally, no differences in regards of total number of Tcells generated (30AE9±3AE1, 29AE7±1AE5 millions⁄ml), foldexpansion (309AE4±31AE8, 297AE4±15AE2) or expression ofCD25, CD27 or CD26L were observed when T cells were cul-tured in RPMI-1640 supplemented with PI-HS or C-HS.Students t-test was used to evaluate the results between PIand control.The presented technique for PI of human serum providesa solution to an important safety and regulatory problem inmodern cell therapy. PI exerts no negative impact onhuman islets of Langerhans, MSCs, T cells or cell lines andcan even have a positive effect by down-regulation ofinflammatory mediators induced by DNA or RNA strandsreleased from damaged cells. INTERCEPT treatment ofhuman serum allows the routine use of human serum inclinical cell transplantation.In many European countries, the Intercept technology isroutinely used for PI of platelets and plasma for clinicaluse. Applying this already used technique in a new waycould be a new niche for blood banks. They can use this toprovide safer products for clinical cell culture and trans-plantation and by doing this increase the safety for thepatients.

Human islet isolation processing times shortened by one hour : minimized incubation time between tissue harvest and islet purification

Human Islet Isolation Processing Times Shortened By One Hour : Minimized Incubation Time Between Tissue Harvest and Islet Purification

Evaluation of Perfluorohexyloctane/Polydimethylsiloxane for Pancreas Preservation for Clinical Islet Isolation and Transplantation

This study aimed to evaluate a 50:50 mix of perfluorohexyloctane/polydimethylsiloxane 5 (F6H8S5) preservation of pancreases in a clinical setting compared with standard solutions for 1) cold ischemia time (CIT) <10 h and 2) an extended CIT >20 h. Procured clinical-grade pancreases were shipped in either F6H8S5 or in standard preservation solutions, that is, University of Wisconsin (UW) or Custodiol. F6H5S5 was preoxygenated for at least 15 min. Included clinical-grade pancreases were procured in UW or Custodiol. Upon arrival at the islet isolation laboratory, the duodenum was removed followed by rough trimming while F6H8S5 was oxygenated for 15-20 min. Trimmed pancreases were immersed into oxygenated F6H8S5 and stored at 4°C overnight followed by subsequent islet isolation. Pancreas preservation using F6H8S5 proved as effective as UW and Custadiol when used within CIT up to 10 h, in terms of both isolation outcome and islet functionality. Preservation in F6H8S5 of pancreases with extended CIT gave results similar to controls with CIT <10 h for both isolated islet functionality and isolation outcome. This study of clinically obtained pancreases indicates a clear benefit of using F6H8S5 on pancreases with extended CIT as it seems to allow extended cold ischemic time without affecting islet function and islet numbers.

Calcium: A Crucial Potentiator for Efficient Enzyme Digestion of the Human Pancreas

BACKGROUND Effective digestive enzymes are crucial for successful islet isolation. Supplemental proteases are essential because they synergize with collagenase for effective pancreatic digestion. The activity of these enzymes is critically dependent on the presence of Ca2+ ions at a concentration of 5-10 mM. The present study aimed to determine the Ca2+ concentration during human islet isolation and to ascertain whether the addition of supplementary Ca2+ is required to maintain an optimal Ca2+ concentration during the various phases of the islet isolation process. METHODS Human islets were isolated according to standard methods and isolation parameters. Islet quality control and the number of isolations fulfilling standard transplantation criteria were evaluated. Ca2+ was determined by using standard clinical chemistry routines. Islet isolation was performed with or without addition of supplementary Ca2+ to reach a Ca2+ of 5 mM. RESULTS Ca2+ concentration was markedly reduced in bicarbonate-based buffers, especially if additional bicarbonate was used to adjust the pH as recommended by the Clinical Islet Transplantation Consortium. A major reduction in Ca2+ concentration was also observed during pancreatic enzyme perfusion, digestion, and harvest. Additional Ca2+ supplementation of media used for dissolving the enzymes and during digestion, perfusion, and harvest was necessary in order to obtain the concentration recommended for optimal enzyme activity and efficient liberation of a large number of islets from the human pancreas. CONCLUSIONS Ca2+ is to a large extent consumed during clinical islet isolation, and in the absence of supplementation, the concentration fell below that recommended for optimal enzyme activity. Ca2+ supplementation of the media used during human pancreas digestion is necessary to maintain the concentration recommended for optimal enzyme activity. Addition of Ca2+ to the enzyme blend has been implemented in the standard isolation protocols in the Nordic Network for Clinical Islet Transplantation.Background: Effective digestive enzymes are crucial for successful islet isolation. Supplemental proteases are essential because they synergize with collagenase for effective pancreatic digestion. The activity of these enzymes is critically dependent on the presence of Ca2+ ions at a concentration of 5–10 mM. The present study aimed to determine the Ca2+ concentration during human islet isolation and to ascertain whether the addition of supplementary Ca2+ is required to maintain an optimal Ca2+ concentration during the various phases of the islet isolation process. Methods: Human islets were isolated according to standard methods and isolation parameters. Islet quality control and the number of isolations fulfilling standard transplantation criteria were evaluated. Ca2+ was determined by using standard clinical chemistry routines. Islet isolation was performed with or without addition of supplementary Ca2+ to reach a Ca2+ of 5 mM. Results: Ca2+ concentration was markedly reduced in bicarbonate-based buffers, especially if additional bicarbonate was used to adjust the pH as recommended by the Clinical Islet Transplantation Consortium. A major reduction in Ca2+ concentration was also observed during pancreatic enzyme perfusion, digestion, and harvest. Additional Ca2+ supplementation of media used for dissolving the enzymes and during digestion, perfusion, and harvest was necessary in order to obtain the concentration recommended for optimal enzyme activity and efficient liberation of a large number of islets from the human pancreas. Conclusions: Ca2+ is to a large extent consumed during clinical islet isolation, and in the absence of supplementation, the concentration fell below that recommended for optimal enzyme activity. Ca2+ supplementation of the media used during human pancreas digestion is necessary to maintain the concentration recommended for optimal enzyme activity. Addition of Ca2+ to the enzyme blend has been implemented in the standard isolation protocols in the Nordic Network for Clinical Islet Transplantation.

Technical Improvement Of Human Pancreatic Islet Isolation

INTRODUCTION A key factor for successful islet isolation is to place the optimal amount of enzyme into the pancreatic ducts prior to starting digestion of pancreatic glands. To improve this procedure, we introduced novel techniques to identify and repair tissue damage resulting in leakage of collagenase solution. MATERIALS AND METHODS One hundred twelve standardized consecutive islet isolations were for the effects of dye and glue on islet yield, islet function using a perifusion assay, and the possibility of clinical transplantation. One group of pancreata (n = 26) obtained en bloc together with duodenum were carefully detached with ligation of accessory ducts in an isolation unit (WPD group), whereas the pancreata were dissected from the duodenum in the operating room in the other 86 isolations. In 28 of 86 isolations, whole glands were used (WP group), while only the body and tail area were applied in the remaining 58 isolations (PP group). RESULTS Both dye and glue effectively prevented leakage of collagenase from the gland. Both islet yield and success rate were higher with these tools without adverse effects on islet function or collagenase activity. The success rate of isolations and islet yield were significantly higher in the WPD group (P = .02 and .001, respectively). CONCLUSIONS Dye and glue may be useful tools to improve human islet isolation procedures. In addition, the use of the whole pancreas further improves the outcome.