Reduction of Activin A gives rise to comparable expression of key definitive endoderm and mature beta cell markers.

Aim: Cell therapies for diabetes rely on differentiation of stem cells into insulin-producing cells, which is complex and expensive. Our goal was to evaluate production costs and test ways to reduce it. Methods: Cost of Goods (COGs) analysis for differentiation was completed and the effects of replacement or reduction of the most expensive item was tested using qRT-PCR, immunohistochemistry, flow cytometry along with glucose-stimulated insulin release. Results: Activin A (AA) was responsible for significant cost. Replacement with small molecules failed to form definitive endoderm (DE). Reducing AA by 50% did not negatively affect expression of beta cell markers. Conclusion: Reduction of AA concentration is feasible without adversely affecting DE and islet-like cell differentiation, leading to significant cost savings in manufacturing.

Controlled-Release Hydrogel Microspheres to Deliver Multipotent Stem Cells for Treatment of Knee Osteoarthritis.

Osteoarthritis (OA) is the most common form of joint disease affecting articular cartilage and peri-articular tissues. Traditional treatments are insufficient, as they are aimed at mitigating symptoms. Multipotent Stromal Cell (MSC) therapy has been proposed as a treatment capable of both preventing cartilage destruction and treating symptoms. While many studies have investigated MSCs for treating OA, therapeutic success is often inconsistent due to low MSC viability and retention in the joint. To address this, biomaterial-assisted delivery is of interest, particularly hydrogel microspheres, which can be easily injected into the joint. Microspheres composed of hyaluronic acid (HA) were created as MSC delivery vehicles. Microrheology measurements indicated that the microspheres had structural integrity alongside sufficient permeability. Additionally, encapsulated MSC viability was found to be above 70% over one week in culture. Gene expression analysis of MSC-identifying markers showed no change in CD29 levels, increased expression of CD44, and decreased expression of CD90 after one week of encapsulation. Analysis of chondrogenic markers showed increased expressions of aggrecan (ACAN) and SRY-box transcription factor 9 (SOX9), and decreased expression of osteogenic markers, runt-related transcription factor 2 (RUNX2), and alkaline phosphatase (ALPL). In vivo analysis revealed that HA microspheres remained in the joint for up to 6 weeks. Rats that had undergone destabilization of the medial meniscus and had overt OA were treated with empty HA microspheres, MSC-laden microspheres, MSCs alone, or a control vehicle. Pain measurements taken before and after the treatment illustrated temporarily decreased pain in groups treated with encapsulated cells. Finally, the histopathological scoring of each group illustrated significantly less OA damage in those treated with encapsulated cells compared to controls. Overall, these studies demonstrate the potential of using HA-based hydrogel microspheres to enhance the therapeutic efficacy of MSCs in treating OA.

PEGDA microencapsulated allogeneic islets reverse canine diabetes without immunosuppression.

BACKGROUND: Protection of islets without systemic immunosuppression has been a long-sought goal in the islet transplant field. We conducted a pilot biocompatibility/safety study in healthy dogs followed by a dose-finding efficacy study in diabetic dogs using polyethylene glycol diacrylate (PEGDA) microencapsulated allogeneic canine islets. METHODS: Prior to the transplants, characterization of the canine islets included the calculations determining the average cell number/islet equivalent. Following measurements of purity, insulin secretion, and insulin, DNA and ATP content, the islets were encapsulated and transplanted interperitoneally into dogs via a catheter, which predominantly attached to the omentum. In the healthy dogs, half of the microspheres injected contained canine islets, the other half of the omentum received empty PEGDA microspheres. RESULTS: In the biocompatibility study, healthy dogs received increasing doses of cells up to 1.7 M cells/kg body weight, yet no hypoglycemic events were recorded and the dogs presented with no adverse events. At necropsy the microspheres were identified and described as clear with attachment to the omentum. Several of the blood chemistry values that were abnormal prior to the transplants normalized after the transplant. The same observation was made for the diabetic dogs that received higher doses of canine islets. In all diabetic dogs, the insulin required to attempt to control blood glucose was cut by 50-100% after the transplant, down to no required insulin for the course of the 60-day study. The dogs had no adverse events and behavioral monitoring suggested normal activity after recovery from the transplant. CONCLUSIONS AND IMPLICATIONS: The study provides evidence that PEGDA microencapsulated canine islets reversed the signs of diabetes without immunosuppression and led to states of insulin-independence or significantly lowered insulin requirements in the recipients.

Viability, yield and expansion capability of feline MSCs obtained from subcutaneous and reproductive organ adipose depots.

BACKGROUND: The source of multipotent stromal cells (MSC) can have a significant influence on the health and expansion capacity of the cells. As the applications for allogeneic MSCs in the treatment of feline diseases increase, the location of the initial donor tissue must be analyzed. To date, comparisons have only been made between feline MSCs collected from bone marrow or abdominal fat. This is the first report to compare cells obtained from different adipose depots in the cat with a focus on clinically relevant donor tissues. The tissue was collected from 34 healthy cats undergoing spaying (fat around the ovaries and uterine horn) or subcutaneous fat collected during surgical procedures. RESULTS: The amount of starting material is essential to isolate sufficient MSCs. The total tissue yield from the subcutaneous fat was significantly greater than could be obtained from around the reproductive organs, leading to 3 times more MSCs per donor. However, the concentration of MSCs obtained from reproductive fat was higher than from subcutaneous fat. In addition, the viability of the MSCs from the reproductive fat was significantly higher than the subcutaneous fat. Since most spaying occurs in young cats (under 18 months) reproductive fat was collected from adult cats during spaying, illustrating that age did not alter the yield or viability of the MSCs. When sufficient tissue was collected, it was digested either mechanically or enzymatically. Mechanical digestion further decreased the viability and yield of MSCs from subcutaneous fat compared to enzymatic digestion. Biomarkers of stem cell characterization, expansion capacity and function were detected using qPCR. CD70, CD90 and CD105 were all expressed in high levels in the 3 groups. However, the reproductive fat had higher levels of CD73 with the mechanically digested subcutaneous fat having the least. Gata6 was detected in all samples while Sox2 and Sox17 were also detected with higher quantities found in the enzymatically digested subcutaneous fat. Negative control genes of Gata4 and Pdx1 showed no detection prior to 50 cycles. During the first three passages, age of the donor, location of the donor tissue, or digestion protocol had no effect on cell culture doubling times or cell viability. CONCLUSIONS: While MSCs from reproductive fat had superior cells/tissue weight and initial viability, there were still dramatically fewer cells obtained compared to subcutaneous fat due to the limited amount of tissue surrounding the reproductive organs. Further, in P1-P3 cultures there were no differences noted in doubling time or cell viability between tissue obtained from reproductive or subcutaneous fat depots.

Hyaluronic Acid Hydrogel Microspheres for Slow Release Stem Cell Delivery.

Cell therapies are hampered by a lack of available delivery systems, resulting in inconsistent outcomes in animal studies and human clinical trials. Hydrogel encapsulants offer a broad range of tunable characteristics in the design of cell delivery vehicles. The focus of the hydrogel field has been on durable encapsulants that provide long-term paracrine function of the cells. However, some cell therapies require cell-to-cell contact in order to elicit their effect. Controlled release microencapsulants would be beneficial in these situations, but appropriate polymers have not been adaptable to microsphere manufacturing because they harden too slowly. We developed and tested a novel microencapsulant formulation (acrylated hyaluronic acid: AHA) with degradation characteristics as a controlled release cell delivery vehicle. The properties of AHA microspheres were evaluated and compared to those of poly(ethylene glycol) diacrylate (PEGDA), a durable hydrogel. AHA microspheres possessed a higher swelling ratio, lower diffusion barrier, faster degradation rate, a lower storage modulus, and a larger average diameter than microspheres composed of PEGDA. Additionally, in vitro cell viability and release and short-term in vivo biocompatibility in immune competent Sprague-Dawley rats was assessed for each microsphere type. Compared to PEGDA, microspheres composed of AHA resulted in significantly less foreign body response in vivo as measured by a lack of cellularity or fibrotic ring in the surrounding tissue and no cellular infiltration into the microsphere. This study illustrates the potential of AHA microspheres as a degradable cell delivery system with superior encapsulated cell viability and biocompatibility with the surrounding tissue.

A Versatile Microencapsulation Platform for Hyaluronic Acid and Polyethylene Glycol.

Cell microencapsulation is a rapidly expanding field with broad potential for stem cell therapies and tissue engineering research. Traditional alginate microspheres suffer from poor biocompatibility, and microencapsulation of more advanced hydrogels is challenging due to their slower gelation rates. We have developed a novel, noncytotoxic, nonemulsion-based method to produce hydrogel microspheres compatible with a wide variety of materials, called core-shell spherification (CSS). Fabrication of microspheres by CSS derived from two slow-hardening hydrogels, hyaluronic acid (HA) and polyethylene glycol diacrylate (PEGDA), was characterized. HA microspheres were manufactured with two different crosslinking methods: thiolation and methacrylation. Microspheres of methacrylated HA (MeHA) had the greatest swelling ratio, the largest average diameter, and the lowest diffusion barrier. In contrast, PEGDA microspheres had the smallest diameters, the lowest swelling ratio, and the highest diffusion barrier, while microspheres of thiolated HA had characteristics that were in between the other two groups. To test the ability of the hydrogels to protect cells, while promoting function, diabetic NOD mice received intraperitoneal injections of PEGDA or MeHA microencapsulated canine islets. PEGDA microspheres reversed diabetes for the length of the study (up to 16 weeks). In contrast, islets encapsulated in MeHA microspheres at the same dose restored normoglycemia, but only transiently (3-4 weeks). Nonencapsulated canine islet transplanted at the same dose did not restore normoglycemia for any length of time. In conclusion, CSS provides a nontoxic microencapsulation procedure compatible with various hydrogel types.

Improved harmonization of critical characterization assays across cell therapies.

The field of cell therapy has blossomed, providing exciting new options for treating a variety of diseases. While few cell therapy products have US FDA approval, there are thousands of cell treatments at various stages of development, pointing to a potential revolutionary shift in patient care. The expanding number and nature of cellular therapies necessitate greater standardization. Several international organizations are collaborating to pursue some level of global standardization, especially concerning cell banking. However, less harmonization surrounds assays used for critical quality characterization including: identity, purity, safety and potency. Frequently, there is divergence regarding the terms describing the characterization assays across regulatory authorities and guidances. This review summarizes the critical quality assays currently used for different categories of cell therapies. Areas of harmonization and an absence of standardization are highlighted. We propose potential solutions to facilitate harmonization of critical quality characterization assays and the language used to describe them.

The Flaws and Future of Islet Volume Measurements.

When working with isolated islet preparations, measuring the volume of tissue is not a trivial matter. Islets come in a large range of sizes and are often contaminated with exocrine tissue. Many factors complicate the procedure, and yet knowledge of the islet volume is essential for predicting the success of an islet transplant or comparing experimental groups in the laboratory. In 1990, Ricordi presented the islet equivalency (IEQ), defined as one IEQ equaling a single spherical islet of 150 µm in diameter. The method for estimating IEQ was developed by visualizing islets in a microscope, estimating their diameter in 50 µm categories and calculating a total volume for the preparation. Shortly after its introduction, the IEQ was adopted as the standard method for islet volume measurements. It has helped to advance research in the field by providing a useful tool improving the reproducibility of islet research and eventually the success of clinical islet transplants. However, the accuracy of the IEQ method has been questioned for years and many alternatives have been proposed, but none have been able to replace the widespread use of the IEQ. This article reviews the history of the IEQ, and discusses the benefits and failings of the measurement. A thorough evaluation of alternatives for estimating islet volume is provided along with the steps needed to uniformly move to an improved method of islet volume estimation. The lessons learned from islet researchers may serve as a guide for other fields of regenerative medicine as cell clusters become a more attractive therapeutic option.

Deletion of the insulin receptor in sensory neurons increases pancreatic insulin levels.

Insulin is known to have neurotrophic properties and loss of insulin support to sensory neurons may contribute to peripheral diabetic neuropathy (PDN). Here, genetically-modified mice were generated in which peripheral sensory neurons lacked the insulin receptor (SNIRKO mice) to determine whether disrupted sensory neuron insulin signaling plays a crucial role in the development of PDN and whether SNIRKO mice develop symptoms of PDN due to reduced insulin neurotrophic support. Our results revealed that SNIRKO mice were euglycemic and never displayed significant changes in a wide range of sensorimotor behaviors, nerve conduction velocity or intraepidermal nerve fiber density. However, SNIRKO mice displayed elevated serum insulin levels, glucose intolerance, and increased insulin content in the islets of Langerhans of the pancreas. These results contribute to the growing idea that sensory innervation of pancreatic islets is key to regulating islet function and that a negative feedback loop of sensory neuron insulin signaling keeps this regulation in balance. Our results suggest that a loss of insulin receptors in sensory neurons does not lead to peripheral nerve dysfunction. The SNIRKO mice will be a powerful tool to investigate sensory neuron insulin signaling and may give a unique insight into the role that sensory neurons play in modifying islet physiology.

Improved yield of canine islet isolation from deceased donors.

BACKGROUND: Canine diabetes is a strikingly prevalent and growing disease, and yet the standard treatment of a twice-daily insulin injection is both cumbersome to pet owners and only moderately effective. Islet transplantation has been performed with repeated success in canine research models, but has unfortunately not been made available to companion animals. Standard protocols for islet isolation, developed primarily for human islet transplantation, include beating-heart organ donation, vascular perfusion of preservation solutions, specialized equipment. Unfortunately, these processes are prohibitively complex and expensive for veterinary use. The aim of the study was to develop a simplified approach for isolating canine islets that is compatible with the financial and logistical restrictions inherent to veterinary medicine for the purpose of translating islet transplantation to a clinical treatment for canine diabetes. RESULTS: Here, we describe simplified strategies for isolating quality islets from deceased canine donors without vascular preservation and with up to 90 min of cold ischemia time. An average of more than 1500 islet equivalents per kg of donor bodyweight was obtained with a purity of 70% (N = 6 animals). Islets were 95% viable and responsive to glucose stimulation for a week. We found that processing only the body and tail of the pancreas increased isolation efficiency without sacrificing islet total yield. Islet yield per gram of tissue increased from 773 to 1868 islet equivalents when the head of the pancreas was discarded (N = 3/group). CONCLUSIONS: In summary, this study resulted in the development of an efficient and readily accessible method for obtaining viable and functional canine islets from deceased donors. These strategies provide an ethical means for obtaining donor islets.

Integration of mesenchymal stem cells into islet cell spheroids improves long-term viability, but not islet function.

Pancreatic islets, especially the large islets (> 150µm in diameter) have poor survival rates in culture. Co-culturing with mesenchymal stem cells (MSCs) has been shown to improve islet survival and function. However, most co-culture studies have been comprised of MSC surrounding islets in the media. The purpose of this study was to determine whether islet survival and function was improved when the 2 populations of cells were intermingled with each other in a defined geometry. Hybrid spheroids containing 25, 50 or 75 or 90% islets cells with appropriate numbers of MSCs were created along with spheroids comprised of only islet cells or only MSCs. Spheroids were tested for yield, viability, diameter, cellular composition, and glucose-stimulated insulin secretion. The 25% islet/75% MSC group created the fewest spheroids, with the poorest survival and insulin secretion and the largest diameter. The remaining groups were highly viable with average diameters under 80µm at formation. However, the hybrid spheroid groups preferred to cluster in islet-only spheroids. The 50, 75 and 90% islet cell groups had excellent long-term survival with 90-95% viability at 2 weeks in culture, compared with the islet only group that were below 80% viability. The glucose-stimulated insulin secretion was not statistically different for the 50, 75, or 90 groups when exposed to 2.4, 16.8, or 22.4 mM glucose. Only the spheroids with 25% islet cells had a statistically lower levels of insulin release, and the 100% had statistically higher levels at 22.4 mM glucose and in response to secretagogue. Thus, imbedded co-culture improved long-term viability, but failed to enhance glucose-stimulated insulin secretion in vitro.

Long-term cryopreservation of reaggregated pancreatic islets resulting in successful transplantation in rats.

Preservation of pancreatic islets for long-term storage of islets used for transplantation or research has long been a goal. Unfortunately, few studies on long-term islet cryopreservation (1 month and longer) have reported positive outcomes in terms of islet yield, survival and function. In general, single cells have been shown to tolerate the cryopreservation procedure better than tissues/multicellular structures like islets. Thus, we optimized a method to cryopreserve single islet cells and, after thawing, reaggregated them into islet spheroids. Cryopreserved (CP) single human islet cells formed spheroids efficiently within 3-5 days after thawing. Approximately 79% of islet cells were recovered following the single-cell cryopreservation protocol. Viability after long-term cryopreservation (4 weeks or more) was significantly higher in the CP islet cell spheroids (97.4 ± 0.4%) compared to CP native islets (14.6 ± 0.4%). Moreover, CP islet cell spheroids had excellent viability even after weeks in culture (88.5 ± 1.6%). Metabolic activity was 4-5 times higher in CP islet cell spheroids than CP native islets at 24 and 48 h after thawing. Diabetic rats transplanted with CP islet cell spheroids were normoglycemic for 10 months, identical to diabetic rats transplanted with fresh islets. However, the animals receiving fresh islets required a higher volume of transplanted tissue to achieve normoglycemia compared to those transplanted with CP islet cell spheroids. By cryopreserving single cells instead of intact islets, we achieved highly viable and functional islets after thawing that required lower tissue volumes to reverse diabetes in rats.

An Automated Multiplexed Hepatotoxicity and CYP Induction Assay Using HepaRG Cells in 2D and 3D.

Drug-induced liver injury (DILI) and drug-drug interactions (DDIs) are concerns when developing safe and efficacious compounds. We have developed an automated multiplex assay to detect hepatotoxicity (i.e., ATP depletion) and metabolism (i.e., cytochrome P450 1A [CYP1A] and cytochrome P450 3A4 [CYP3A4] enzyme activity) in two-dimensional (2D) and three-dimensional (3D) cell cultures. HepaRG cells were cultured in our proprietary micromold plates and produced spheroids. HepaRG cells, in 2D or 3D, expressed liver-specific proteins throughout the culture period, although 3D cultures consistently exhibited higher albumin secretion and CYP1A/CYP3A4 enzyme activity than 2D cultures. Once the spheroid hepatic quality was assessed, 2D and 3D HepaRGs were challenged to a panel of DILI- and CYP-inducing compounds for 7 days. The 3D HepaRG model had a 70% sensitivity to liver toxins at 7 days, while the 2D model had a 60% sensitivity. In both the 2D and 3D HepaRG models, 83% of compounds were predicted to be CYP inducers after 7 days of compound exposure. Combined, our results demonstrate that an automated multiplexed liver spheroid system is a promising cell-based method to evaluate DILI and DDI for early-stage drug discovery.

Hyaluronic Acid/Collagen Hydrogel as an Alternative to Alginate for Long-Term Immunoprotected Islet Transplantation.

Alginate has long been the material of choice for immunoprotection of islets due to its low cost and ability to easily form microspheres. Unfortunately, this seaweed-derived material is notoriously prone to fibrotic overgrowth in vivo, resulting in premature graft failure. The purpose of this study was to test an alternative, hyaluronic acid (HA-COL), for in vitro function, viability, and allogeneic islet transplant outcomes in diabetic rats. In vitro studies indicated that the HA-COL gel had diffusion characteristics that would allow small molecules such as glucose and insulin to enter and exit the gel, whereas larger molecules (70 and 500 kDa dextrans) were impeded from diffusing past the gel edge in 24 h. Islets encapsulated in HA-COL hydrogel showed significantly improved in vitro viability over unencapsulated islets and retained their morphology and glucose sensitivity for 28 days. When unencapsulated allogeneic islet transplants were administered to the omentum of outbred rats, they initially were normoglycemic, but by 11 days returned to hyperglycemia. Immunohistological examination of the grafts and surrounding tissue indicated strong graft rejection. By comparison, when using the same outbred strain of rats, allogeneic transplantation of islets within the HA-COL gel reversed long-term diabetes and prevented graft rejection in all animals. Animals were sacrificed at 40, 52, 64, and 80 weeks for evaluation, and all were non-diabetic at sacrifice. Explanted grafts revealed viable islets in the transplant site as well as intact hydrogel, with little or no evidence of fibrotic overgrowth or cellular rejection. The results of these studies demonstrate great potential for HA-COL hydrogel as an alternative to sodium alginate for long-term immunoprotected islet transplantation.

A simple, reliable method for high-throughput screening for diabetes drugs using 3D ß-cell spheroids.

Early screens for new diabetes drugs rely on monolayers of ß-cells, which are known to be poor predictors of the in vivo response. Previously, we developed a method to create uniform islet spheroids from freshly-dispersed human donor tissue for drug screening. While the human engineered islets worked well to reduce donor-to-donor variability, it is difficult and expensive to obtain sufficient high-quality human islets for drug testing. Thus, this study utilized a genetically-modified ß-cell culture line (INS-1832/13) in 2D and as 3D spheroids and compared the results to human islet tissue formed into spheroids using a high-throughput 384-well format. In response to increasing concentrations of glucose, all 3 groups increased insulin release, but the cultured ß-cells (2D and 3D) were more sensitive to glucose (EC50 5.85mM for 2D ß-cells, 16.24mM for 3D ß-cell spheroids) than the human islet spheroids (EC50 53.69mM). The order of responses to glybenclamide was human spheroids >3D ß-cell culture >2D ß-cell culture. In response to caffeine, the ß-cells in 2D or 3D were more responsive compared to the human islet spheroids (EC50 0.39 and 0.31mM for 2D and 3D ß-cells respectively). When exposed to inhibitors of insulin secretion (nifedipine and diazoxide), the responses were more similar between groups. Z' calculations, indicative of assay quality, determined that the 3D ß-cell spheroids reached the criteria of an excellent to ideal drug screen assay more consistently than the other test models. In conclusion, 3D ß-cell spheroids from a cultured cell line can be used in HTS assays that, according to reference drugs tested here, are sensitive and predictive of the in vivo response.

Arum Palaestinum with isovanillin, linolenic acid and ß-sitosterol inhibits prostate cancer spheroids and reduces the growth rate of prostate tumors in mice.

BACKGROUND: Arum palaestinum is a plant commonly found in the Middle East that is ingested as an herbal remedy to fight cancer. However, no studies have examined the direct effect of the plant/plant extract on tumor growth in an animal model. METHODS: Verified prostate cancer cells were plated as 3D spheroids to determine the effect of extract from boiled Arum Palaestinum Boiss roots. In addition, male NU/NU mice (8 weeks old) with xenograft tumors derived from the prostate cancer cell line were treated daily with 1000 mg/kg body weight gavage of the suspension GZ17. The tumor growth was measured repeatedly with calipers and the excised tumors were weighed at the termination of the 3 week study. Control mice (10 mice in each group) received vehicle in the same manner and volume. RESULTS: The number of live prostate cancer cells declined in a dose/dependent manner with a 24 h exposure to the extract at doses of 0.015 to 6.25 mg/mL. A fortified version of the extract (referred to as GZ17) that contained higher levels of isovanillin, linolenic acid and ß-sitosterol had a stronger effect on the cell death rate, shifting the percentage of dead cells from 30 % to 55 % at the highest dose while the vehicle control had no effect on cell numbers. When GZ17 was applied to non-cancer tissue, in this case, human islets, there was no cell death at doses that were toxic to treated cancer cells. Preliminary toxicity studies were conducted on rats using an up-down design, with no signs of toxic effect at the highest dose. NU/NU mice with xenograft prostate tumors treated with GZ17 had a dramatic inhibition of tumor progression, while tumors in the control group grew steadily through the 3 weeks. The rate of tumor volume increase was 73 mm(3)/day for the vehicle group and 24 mm(3)/day for the GZ17 treated mice. While there was a trend towards lower excised tumor weight at study termination in the GZ17 treatment group, there was no statistical difference. CONCLUSIONS: Fortified Arum palaestinum Boiss caused a reduction in live cells within prostate cancer spheroids and blocked tumor growth in xenografted prostate tumors in mice without signs of toxicity.

A Simple Method to Replace Islet Equivalents for Volume Quantification of Human Islets.

Human islets come in a variety of sizes and shapes, and the total volume of islets used for research or clinical transplants must be estimated in a manner that is simple and valid. Islet equivalent (IEQ) measurements are the standard estimate of islet volume. We published a new method (the Kansas method) for estimating rat islet volume using cell numbers that was reliable and valid. Here we modified the method for human islets. We measured the dimensions of isolated human islets showing that they are not spherical and became less so in larger islets, with an average smallest/largest diameter ratio of 0.73 in large islets and 0.85 in small islets. Human islets were individually loaded into 96-well plates, dissociated into single cells, and the total cell number per islet determined with computer-assisted cytometry. Based on the counted cell number per islet, a regression model was created to convert islet diameter to cell number with a high R(2) value (0.99). Separate regression equations for male and female donors or young and old donors were not significantly different than the pooled data and did not improve the regression values. There was an inverse correlation between the cell number per IEQ and islet size. The Kansas method was validated with ATP/cell and cell viability data. Compared to the actual cell count, conventional IEQ measurements overestimated tissue volume of large islets by nearly double. Examples of differences in results obtained from the same data sets normalized to IEQ or the Kansas method included viability and insulin secretion concentrations. The implications of the error associated with the current IEQ method of volume estimation are discussed.

Differences in insulin biosynthesis pathway between small and large islets do not correspond to insulin secretion.

In a variety of mammalian species, small islets secrete more insulin per volume than large islets. This difference may be due to diffusional limitations of large islets, or inherent differences in the insulin production pathways. The purpose of this study was to identify possible differences in the early phase of glucose-stimulated insulin biosynthesis between large and small islets. Isolated small and large rat islets were challenged with 30 minutes of high glucose. The expression of insulin gene transcription factors (MafA, NeuroD/ Beta2, and PDX-1), preproinsulin mRNA, proinsulin and insulin were compared between large and small islets. Under basal (low glucose) conditions, MafA and NeuroD had higher mRNA levels and greater protein amounts in large islets compared to small when normalized to GAPDH levels. 30 minutes of high glucose stimulation failed to alter the mRNA or subsequent protein levels of either gene. However, 30 minutes of high glucose suppressed activated PDX-1 protein levels in both small and large islets. High glucose stimulation did not statistically alter the preproinsulin mRNA (insulin 1 and insulin 2) levels. At the translational level, high glucose increased the proinsulin levels, and large islets showed a higher proinsulin content per cell than small islets. Insulin content per cell was not significantly different between small and large islets under basal or high glucose levels. The results fail to explain the higher level of insulin secretion noted in small versus large islets and may suggest that possible differences lie downstream in the secretory pathway rather than insulin biosynthesis.