Transport Limitations in Islets of Langerhans Culture

Introduction: Islet transplantation has become a promising treatment for type I diabetes mellitus due to recent success since the development of the Edmonton Protocol. Islet culture prior to transplantation is standard practice in several clinical islet programs. High-density islet culture is desirable because it reduces space and handling requirements during culture, but it exacerbates oxygen limitations and causing a reduction in islet viability. We investigated the effect of tissue density on total tissue recovery, viable tissue recovery, and tissue purity for conventional normoxic culture on a polystyrene dish. To improve islet quality in high density culture, we explored use of elevated pO2 or culture on an oxygen-permeable silicone rubber membrane. We applied a theoretical O2 transport model to investigate how O2 transport changes for each culture condition and then compared our predictions to the experimental data to determine whether O2 is limiting during high density culture using these new techniques. Methods: Human islets were cultured for 34-60 hr at densities varying from 20-5300 IE/cm on either 500-μm silicone membranes or solid bottom dishes in a humidified incubator with 5% CO2, 19% or 56% oxygen at 37oC. The quantity, viability, and purity of tissue were quantified using nuclei counts, oxygen consumption rate (OCR) measurements, and insulin immunostaining. Theoretically predicted profiles of oxygen partial pressure and islet viability were obtained by solution of the species conservation equation with the finite element method. Results: Low density islet culture on a polystyrene dish resulted in tissue adherence, which decreased as density increased. The adherent tissue had a lower fraction of original insulinpositive cells collected from culture as compared to the non-adherent tissue. The combined non-adherent and adherent tissue collected from low density culture accounted for nearly 100% of the original viable tissue placed into culture and had purity similar to that for tissue prior to culture. With conventional culture, recovery of total OCR decreased sharply as viable tissue density increased. At all densities, the fraction of original viable tissue collected from culture was higher with culture on polystyrene dishes in 56% oxygen and even higher with normoxic culture on silicone rubber. Theoretical predictions were qualitatively similar to experimental results but in general over predicted the amount of viable tissue recovered. Conclusions: In low density culture all original viable tissue can be recovered as both free and adherent tissue. In high density culture, recovery of viable tissue (1) decreases as culture density increases on a polystyrene surface, (2) increases with increasing external pO2, and (3) increases substantially with culture on silicone rubber by removing O2 limitations. Introduction Though many complications remain [1], islet transplantation is beginning to become a promising option for treating type 1 diabetes due to recent success since the development of the Edmonton Protocol [2]. After islet tissue is mechanically and enzymatically isolated from a donor pancreas [3, 4] it may transplanted directly or cultured for a period of time prior to transplantation [4, 5]. Islet culture prior to transplantation is desirable for many reasons and is standard practice in several clinical islet programs [5]. Culture provides time for islet tissue to recover from the harsh isolation process and allows islets to be maintained over time while tissue quality is assessed for purity, viability, and sterility [5-7]. Islet culture has been the subject of much research but remains an area that needs improvement. Conditions including islet density, culture temperature, and culture duration vary between islet centers with relatively little published justification making it difficult to determine which technique is optimal for islet recovery, viability, and purity, yet still practical in a clinical setting. Though a lot of literature is available investigating various media formulations and additives (see Murdoch et al [5] and Clayton et al [7] for comprehensive reviews on media supplementation), very little is available investigating oxygen (O2) transport and the effect of islet density on islet quality [5]. Gaber et al [8] noted that islet density and size might be related to necrotic death and then Matsumoto et al [6] demonstrated that low islet concentrations resulted in higher recoveries and higher stimulation indexes compared to culture with high islet concentrations. This decrease in tissue quality is believed to be due to insufficient nutrient transport to the core of the islet, which is supported by observations that indicate higher necrotic death in islets at 37°C compared to lower temperatures where metabolic rates are reduced [9]. Dionne et al [10] noted that transport is crucial for maintaining islet viability and function. Since blood vessels collapse during static culture [11, 12], O2 is supplied to the islets only through diffusion resulting in the formation of O2 gradients in and around each islet. If islets are too large or too close together, the O2 is depleted before it reaches the center of the islet causing the core to become hypoxic and leading to the development of a necrotic core [9, 10, 13]. A uniform surface coverage of no more than 0.4% or 21 IE/cm in 3 mm of medium at 37 ̊C and an atmosphere of 95% air/5% CO2 is needed to remove all O2 limitations during culture [13]. This low-density culture requires large space and handling requirements. Assume that 400,000 islets are obtained from an isolation that have to be cultured prior to transplantation. Culture at 21 IE/cm would require an area of about 1.9 m for 400,000 islets. Using T-175 (surface area 175 cm) T-flasks would require use of 109 flasks in order to remove all O2 limitations. This would require 2-3 incubators and someone to consolidate all 109 flasks prior to transplantation which is impractical for clinical use. As a result a tissue density is chosen that is practical at the expense of the viable tissue. Current techniques include culture densities up to 400 IE/cm, which based on previous predictions could result in a 20% loss in viable tissue [13]. Thus, a high density culture technique that maintains tissue quality is desirable in order to keep tissue densities practical without sacrificing viable tissue. This study will focus on two methods for alleviating O2 limitations in high density culture. They are culture in elevated gas pO2 and culture on silicone rubber membranes. In this study we investigate the effect of tissue density on the fraction of original tissue collected from culture, the fraction of original viable tissue collected from culture, and the fraction of original insulin positive cells collected from culture for conventional culture on a polystyrene dish. We explore high density culture techniques by culturing islets either in an elevated pO2 or on an O2-permeable silicone rubber membrane. Finally we use a theoretical O2 transport model to investigate how O2 transport changes for each culture condition and then compare our predictions to the experimental data to determine whether O2 is limiting during high density culture using these new techniques. Methods Islet Culture. Upon receipt a portion of the human islet sample was analyzed and the remainder was placed into culture on either untreated polystyrene culture vessels or 500 μm thick silicone rubber membranes (Wilson Wolf Manufacturing, Inc. New Brighton, MN) at 37°C in a humidified environment with 95% air / 5% CO2 (pO2 = 142 mmHg) at densities varying from 20-5300 IE/cm for 34-60 hr over which no medium change occurred. Islets were cultured in supplemented RPMI (Mediatech Inc., Herndon, VA) with 10% FBS (Mediatech Inc.). For high-density experiments (>30 IE/cm assuming 1560 nuclei/IE) islets were cultured in 0.3-2.2 cm of medium as specified. For low density experiments (<30 IE/cm) islets were cultured in 1.3 mm of medium. For high O2 culture, islets were cultured at 37°C in 56% O2 (428 mmHg). After culture free and adherent tissue were collected and analyzed. For low-density culture, trypsinization was required to remove tissue that adhered to the culture flask. At higher densities trypsinization was not required because tissue did not adhere. Only non-adherent tissue was analyzed at an OCR density > 0.5 nmol/min cm, whereas adherent and nonadherent tissue was combined before analysis when the OCR density was < 0.5 nmol/min cm. Cell Enumeration by Nuclei Counting. Equal volumes of sample and lysis solution were combined, vortexed, and incubated at room temperature. The islet mixture was rapidly forced through a needle to liberate the nuclei. Isolated nuclei were diluted with Dulbecco’s phosphate buffered saline (D-PBS, Invitrogen, Carlsbad, CA), stained with 7-aminoactinomycin D (7-AAD, Molecular Probes, Eugene, OR) for at least 2 min at room temperature, and then analyzed using a flow cytometer (Guava Personal Cell Analysis (PCA) system, Guava Technologies, Hayward, CA). Oxygen Consumption Rate (OCR). Islets were suspended in serum free DMEM (Mediatech Inc) and sealed in a 200-μl stirred titanium chamber (Instech Laboratories, Plymouth Meeting, PA) equipped with a fluorescence-based oxygen sensor (Ocean Optics, Dunedin, FL). The time-dependent pO2 within the chamber was recorded, and the data at high pO2 were fit to a straight line. The maximal OCR was evaluated from OCR = Vchα(dpO2/dt), where Vch is the chamber volume and α is the Bunsen solubility coefficient. Immunostaining and Light Microscopy. Cultured tissue samples were washed twice with DPBS prior to fixing for one hr in 10% formalin (Sigma Aldrich). After fixation the tissue was washed, pelleted in 2% agarose, incubated for 1 hr at room temperature in 10% formalin, and then stored in D-PBS until embedding in paraffin. One-μm sections were taken from the pellet. The sections were stained by immunoperoxidase [14, 15] for β cells as previously described [16]. The primary antibody used in the staining procedure was guinea pig anti-bovine insulin (Linco, St. Charles, MO) and the secondary antibody was goat anti-guinea pig (Cappel, Irvine, CA). The peroxidase-anti-peroxidase (PAP) antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) was applied prior to staining in 50 ml of a 2 mM 3,3’-diaminobenzidine tetrahydrochloride (Sigma Aldrich) solution activated with 25 μl of 30% hydrogen peroxide (EMD Chemicals Inc, Gibbstown, NJ). The slides were counterstained with hematoxylin (Sigma Aldrich). Original tissue samples were analyzed using morphological analysis carried out by light microscopy. Aliquots from the islet preparation were processed using a standard procedure, during which they are fixed, dehydrated, cleared, embedded, cured, and trimmed to produce 1μm sections in epoxy resin. This process took about 3 days. One-μm sections were analyzed by stereological point counting [17] in which point intercepts of tissue with a grid covering adjacent, non-overlapping fields were counted under a light microscope. In order to compare the purity measurement using light microscopy and that with immunoperoxidase insulin staining we converted the volume fraction of islet tissue from light microscopy to the volume fraction β cells excluding vascular space using previously described mathematical relationships [3]. Statistics. Each measurement was run in triplicate unless otherwise specified. Data are expressed as mean ± standard deviation. Statistical significance was determined by a twoway Student t-test for p < 0.05 assuming either paired data. Finite Element Analysis. The finite element simulations using our theoretical model were carried out using the commercially available package COMSOL Multiphysics in conjunction with Matlab. We required the mesh to contain more than 1000 nodes and set a convergence criterion such that the error estimated by COMSOL Multiphysics program, which took into account the current and previous solution in the iterative solver, was less than 10 [18]. Theoretical Model We modeled islet culture on O2-impermeable and O2-permeable dishes assuming the OCR of the tissue follows Michaelis-Menten kinetics. The solution to the diffusion-reaction equation