Modeling ultra-high dose rate electron and proton FLASH effect with the physicochemical approach.

Objective. A physicochemical model built on the radiochemical kinetic theory was recently proposed in (Labarbeet al2020) to explain the FLASH effect. We performed extensive simulations to scrutinize its applicability for oxygen depletion studies and FLASH-related experiments involving both proton and electron beams.Approach. Using the dose and beam delivery parameters for each FLASH experiment, we numerically solved the radiochemical rate equations comprised of a set of coupled nonlinear ordinary differential equations to obtain the area under the curve (AUC) of radical concentrations.Main results. The modeled differences in AUC induced by ultra-high dose rates appeared to correlate well with the FLASH effect. (i) For the whole brain irradiation of mice performed in (Montay-Gruelet al2017), the threshold dose rate values for memory preservation coincided with those at which AUC started to decrease much less rapidly. (ii) For the proton pencil beam scanning FLASH of (Cunninghamet al2021), we found linear correlations between radicals' AUC and the biological endpoints: TGF-ß1, leg contracture and plasma level of cytokine IL-6. (iii) Compatible with the findings of the proton FLASH experiment in (Kimet al2021), we found that radicals' AUC at the entrance and mid-Spread-Out Bragg peak regions were highly similar. In addition, our model also predicted ratios of oxygen depletionG-values between normal and UHDR irradiation similar to those observed in (Caoet al2021) and (El Khatibet al2022).Significance. Collectively, our results suggest that the normal tissue sparing conferred by UHDR irradiation may be due to the lower degree of exposure to peroxyl and superoxide radicals. We also found that the differential effect of dose rate on the radicals' AUC was less pronounced at lower initial oxygen levels, a trait that appears to align with the FLASH differential effect on normal versus tumor tissues.

Multicenter Assessment of Animal-free Collagenase AF-1 for Human Islet Isolation.

Animal-free (AF) SERVA Collagenase AF-1 and Neutral Protease (NP) AF GMP Grade have recently become available for human islet isolation. This report describes the initial experiences of 3 different islet transplant centers. Thirty-four human pancreases were digested using 1 vial of the 6 different lots of Collagenase AF-1 (2,000-2,583 PZ-U/vial) supplemented with 4 different lots of NP AF in a range of 50 to 160 DMC-U per pancreas. Isolation, culture, and quality assessment were performed using standard techniques as previously described. All data are presented as mean ± standard error of the mean (SEM). Variability of pancreas weight was associated with a wide range of collagenase and NP activities, ranging from 12.7 to 46.6 PZ-U/g (26.0 ± 1.5 PZ-U/g) and 0.4 to 3.0 DMC-U/g (1.5 ± 0.1 DMC-U/g), respectively. Postpurification islet yield was 296,494 ± 33,620 islet equivalents (IEQ) equivalent to 3,274 ± 450 IEQ/g with a purity of 55.9% ± 3.2%. Quality assessment performed after 2 to 4 d of culture demonstrated a viability of 88.1% ± 1.5% and a stimulation index of 3.7 ± 0.7. Eighteen of the 34 preparations were transplanted into type 1 diabetic patients equivalent to a transplantation rate of 52.9%. Six preparations, which were infused into patients as first transplant, could be analyzed and increased the fasting C-peptide level from 0.11 ± 0.08 pretransplant to 1.23 ± 0.24 and 2.27 ± 0.31 ng/mL 3 and 6 mo posttransplant ( P < 0.05), respectively. Insulin requirements were simultaneously reduced at the same time from 39.2 ± 3.8 IU/d before transplantation to 10.8 ± 4.1 and 4.0 ± 2.3 IU/d, after 3 and 6 mo posttransplant ( P < 0.05), respectively. This study demonstrates the efficiency of AF SERVA Collagenase AF-1 and NP AF for clinical islet isolation and transplantation. The new plant-based production process makes these products a safe new option for the islet field.

National Institutes of Health-Sponsored Clinical Islet Transplantation Consortium Phase 3 Trial: Manufacture of a Complex Cellular Product at Eight Processing Facilities.

Eight manufacturing facilities participating in the National Institutes of Health-sponsored Clinical Islet Transplantation (CIT) Consortium jointly developed and implemented a harmonized process for the manufacture of allogeneic purified human pancreatic islet (PHPI) product evaluated in a phase 3 trial in subjects with type 1 diabetes. Manufacturing was controlled by a common master production batch record, standard operating procedures that included acceptance criteria for deceased donor organ pancreata and critical raw materials, PHPI product specifications, certificate of analysis, and test methods. The process was compliant with Current Good Manufacturing Practices and Current Good Tissue Practices. This report describes the manufacturing process for 75 PHPI clinical lots and summarizes the results, including lot release. The results demonstrate the feasibility of implementing a harmonized process at multiple facilities for the manufacture of a complex cellular product. The quality systems and regulatory and operational strategies developed by the CIT Consortium yielded product lots that met the prespecified characteristics of safety, purity, potency, and identity and were successfully transplanted into 48 subjects. No adverse events attributable to the product and no cases of primary nonfunction were observed.

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 1520 min. Trimmed pancreases were immersed into oxygenated F6H8S5 and stored at 4C 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.

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

UNLABELLED: 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.

Supplements in human islet culture: human serum albumin is inferior to fetal bovine serum.

Culture of human islets before clinical transplantation or distribution for research purposes is standard practice. At the time the Edmonton protocol was introduced, clinical islet manufacturing did not include culture, and human serum albumin (HSA), instead of fetal bovine serum (FBS), was used during other steps of the process to avoid the introduction of xenogeneic material. When culture was subsequently introduced, HSA was also used for medium supplementation instead of FBS, which was typically used for research islet culture. The use of HSA as culture supplement was not evaluated before this implementation. We performed a retrospective analysis of 103 high-purity islet preparations (76 research preparations, all with FBS culture supplementation, and 27 clinical preparations, all with HSA supplementation) for oxygen consumption rate per DNA content (OCR/DNA; a measure of viability) and diabetes reversal rate in diabetic nude mice (a measure of potency). After 2-day culture, research preparations exhibited an average OCR/DNA 51% higher (p < 0.001) and an average diabetes reversal rate 54% higher (p < 0.05) than clinical preparations, despite 87% of the research islet preparations having been derived from research-grade pancreata that are considered of lower quality. In a prospective paired study on islets from eight research preparations, OCR/DNA was, on average, 27% higher with FBS supplementation than that with HSA supplementation (p < 0.05). We conclude that the quality of clinical islet preparations can be improved when culture is performed in media supplemented with serum instead of albumin.

Transplanted functional islet mass: donor, islet preparation, and recipient factors influence early graft function in islet-after-kidney patients.

BACKGROUND: The ability to predict clinical function of a specific islet batch released for clinical transplantation using standardized variables remains an elusive goal. METHODS: Analysis of 10 donor, 7 islet isolation, 3 quality control, and 6 recipient variables was undertaken in 110 islet-after-kidney transplants and correlated to the pre- to 28-day posttransplant change in C-peptide to glucose and creatinine ratio (ΔCP/GCr). RESULTS: Univariate analysis yielded islet volume transplanted (Spearman r=0.360, P<0.001) and increment of insulin secretion (r=0.377, P<0.001) as variables positively associated to ΔCP/GCr. A negative association to ΔCP/GCr was cold ischemia time (r=-0.330, P<0.001). A linear, backward-selection multiple regression was used to obtain a model for the transplanted functional islet mass (TFIM). The TFIM model, composed of islet volume transplanted, increment of insulin secretion, cold ischemia time, and exocrine tissue volume transplanted, accounted for 43% of the variance of the clinical outcome in the islet-after-kidney data set. CONCLUSION: The TFIM provides a straightforward and potent tool to guide the decision to use a specific islet preparation for clinical transplantation.

Quantification of the islet product: presentation of a standardized current good manufacturing practices compliant system with minimal variability.

BACKGROUND: Accurate islet quantification has proven difficult to standardize in a good manufacturing practices (GMP) approved manner. METHODS: The influence of assessment variables from both manual and computer-assisted digital image analysis (DIA) methods were compared using calibrated, standardized microspheres or islets alone. Additionally, a mixture of microspheres and exocrine tissue was used to evaluate the variability of both the current, internationally recognized, manual method and a novel GMP-friendly purity- and volume-based method (PV) evaluated by DIA in a semiclosed, culture bag system. RESULTS: Computer-assisted DIA recorded known microsphere size distribution and quantities accurately. By using DIA to evaluate islets, the interindividual manually evaluated percent coefficients of variation (CV%; n=14) were reduced by almost half for both islet equivalents (IEs; 31% vs. 17%, P=0.002) and purity (20% vs. 13%, P=0.033). The microsphere pool mixed with exocrine tissue did not differ from expected IE with either method. However, manual IE resulted in a total CV% of 44.3% and a range spanning 258 k IE, whereas PV resulted in CV% of 10.7% and range of 60 k IE. Purity CV% for each method were similar approximating 10.5% and differed from expected by +7% for the manual method and +3% for PV. CONCLUSION: The variability of standard counting methods for islet samples and clinical quantities of microspheres mixed with exocrine tissue were reduced with DIA. They were reduced even further by use of a semiclosed bag system compared with standard manual counting, thereby facilitating the standardization of islet evaluation according to GMP standards.

Large-scale bioreactor expansion of tumor-infiltrating lymphocytes.

BACKGROUND: The aim of this study was to evaluate an improved technique for expansion of tumor-infiltrating lymphocytes (TILs) based on the WAVE Bioreactor system with perfusion and tube-welding techniques. Our hypothesis was that the bioreactor would allow for optimized provision of nutrients and removal of spent media while minimizing culture volumes. These refinements might lead to a better quality of expanded cells with lower amounts of exhausted cells compared to static expansions in culture bags. PROCEDURES: Tumor-infiltrating lymphocytes from 4 melanoma patients were expanded and compared in parallel using either the WAVE Bioreactor 2/10 System or traditional static culture methods. The parameters viability, final cell number, phenotype and effector function were measured. RESULTS: Our results show that the bioreactor system with perfusion is suitable for large-scale expansion of tumor-infiltrating lymphocytes and allows for higher cell densities and absolute cell numbers as compared to static culture conditions. Phenotypic characteristics of TILs were compared pre and post expansion and showed no consistent difference between the two expansion methods. TILs harvested had the phenotype and function corresponding to intermediate to late effector cells. The system allows one technician to operate several bioreactors simultaneously, thereby reducing the labor for one expansion to approximately 1/3 compared to static expansion. DISCUSSION: The WAVE Bioreactor system is suitable for large-scale expansion of TILs. Due to constant perfusion of fresh media and removal of spent media much higher cell densities were achieved while the culture volume and the glucose and glutamine levels were kept constant. Expansion of TILs in the bioreactor system represents a labor- and cost-effective method to reach large numbers of T cells for adoptive cell transfer therapy in the clinic. CONCLUSION: The system presented herein offers an effective alternative to large-scale production of cell products for clinical use while meeting requirements of therapeutic cell quantities and qualities of current protocols for treatment of malignant melanoma.

The importance of tryptic-like activity in purified enzyme blends for efficient islet isolation.

BACKGROUND: The isolation of islets from the human pancreas critically depends on an efficient enzyme blend. Previous studies have solely focused on the presence of collagenase and neutral protease/thermolysin. Despite improved characterization of these components, the lot-related variability in efficacy still persists suggesting that additional so far disregarded enzymes are required for efficient islet cleavage. METHODS: Varying activities of a tryptic-like enzyme were identified within collagenase NB1 lots, which were selected according to a matched ratio between tryptic-like and collagenase activity (TLA-ratio). Rat and human pancreata were processed with current standard procedures. RESULTS: Increasing the TLA-ratio from 1.3% to 10% reduced pancreas dissociation time in rats by 50% without affecting islet yield, viability, or posttransplant function in diabetic nude mice. Enhancing the TLA-ratio from 1.3% to 12.6% for human pancreas processing resulted in a significant reduction of recirculation time and increased incrementally human islet yield without affecting purity, in vitro function or recovery after culture. Optimized pancreas digestion correlated with a higher percentage of islet preparations fulfilling quality criteria for clinical transplantation. CONCLUSIONS: We conclude that TLA is an effective component that should be included in moderate amounts in enzyme blends for human islet isolation to optimize the efficiency and minimize the lot-related variability.