Richard D. Hall

Three-Dimensional Culture of Mouse Renal Carcinoma Cells in Agarose Macrobeads Selects for a Subpopulation of Cells with Cancer Stem Cell or Cancer Progenitor Properties

The culture of tumor cell lines in three-dimensional scaffolds is considered to more closely replicate the in vivo tumor microenvironment than the standard method of two-dimensional cell culture. We hypothesized that our method of encapsulating and maintaining viable and functional pancreatic islets in agarose-agarose macrobeads (diameter 6-8 mm) might provide a novel method for the culture of tumor cell lines. In this report we describe and characterize tumor colonies that form within macrobeads seeded with mouse renal adenocarcinoma cells. Approximately 1% of seeded tumor cells survive in the macrobead and over several months form discrete elliptical colonies appearing as tumor cell niches with increasing metabolic activity in parallel to colony size. The tumor colonies demonstrate ongoing cell turnover as shown by BrdU incorporation and activated caspase-3 and TUNEL staining. Genes upregulated in the tumor colonies of the macrobead are likely adaptations to this novel environment, as well as an amplification of G(1)/S cell-cycle checkpoints. The data presented, including SCA-1 and Oct4 positivity and the upregulation of stem cell-like genes such as those associated with the Wnt pathway, support the notion that the macrobead selects for a subpopulation of cells with cancer stem cell or cancer progenitor properties.

Encapsulation of Porcine Islets Permits Extended Culture Time and Insulin Independence in Spontaneously Diabetic BB Rats

The ability to culture porcine islets for extended times allows for both their functional assessment and the assurance of their microbiological safety prior to transplantation. We have previously shown that agarose-encapsulated porcine islets can be cultured for at least 24 weeks. In the current study, porcine islet agarose macrobeads cultured for up to 67 weeks were assessed for their ability to restore normoglycemia, respond to an intraperitoneal glucose challenge, maintain spontaneously diabetic BB rats free of insulin therapy for more than 6 months, and for their biocompatibility. Porcine islets were encapsulated in agarose macrobeads and subjected to weekly static perifusion assays for the assessment of insulin production. After in vitro culture for either 9, 40, or 67 weeks, 56–60 macrobeads were transplanted to each spontaneously diabetic BB rat. Transplanted rats were monitored daily for blood glucose levels. Glucose tolerance tests and assessments for porcine C-peptide were conducted at various intervals throughout the study. Normoglycemia (100–200 mg/dl) was initially restored in all islet transplanted rats. Moderate hyperglycemia (200–400 mg/dl) developed at around 30 days posttransplantation and continued throughout the study period of 201–202 days. Importantly, all rats that received encapsulated porcine islets continued to gain weight and were free of exogenous insulin therapy for the entire study. Porcine C-peptide (0.2–0.9 ng/ml) was detected in the serum of islet recipients throughout the study period. No differences were detected between recipient animals receiving islet macrobeads of various ages. These results demonstrate that the encapsulation of porcine islets in agarose macrobeads allows for extended culture periods and is an appropriate strategy for functional and microbiological assessment prior to clinical use.

The Use of Pancreas Biopsy Scoring Provides Reliable Porcine Islet Yields While Encapsulation Permits the Determination of Microbiological Safety

For clinical xenogenic islet transplantation to be successful, several requirements must be met. Among them is a sizeable and reliable source of fully functional and microbiologically safe islets. The inherent variability among porcine pancreases, with respect to islet yield, prompted us to develop a Biopsy Score technique to determine the suitability of each pancreas for islet isolation processing. The Biopsy Score consists of an assessment of five variables: warm ischemia time, pancreas color, fat content, islet size, and islet demarcation, each of which is assigned a value of −1 or +1, depending on whether or not the established criteria is met. For determination of islet size and demarcation, fresh biopsies of porcine pancreases are stained with dithizone (DTZ) solution and examined under a dissecting microscope. Based on the scoring of such biopsies in pancreases from 26—56-month-old sows, we report here that the presence of large (>100 μm diameter), well-demarcated islets in the pancreas biopsy is a reliable predictor of isolation success. Encapsulation of the isolated porcine islets within the inner layer of a 1.5% agarose and an outer layer of 5.0% agarose macrobead, containing 500 equivalent islet number (EIN), provides for extended in vitro functional viability (>6 months of insulin production in response to glucose), as well as for comprehensive microbiological testing and at least partial isolation of the xenogeneic islets from the host immune system. All microbiological testing to date has been negative, except for the presence of porcine endogenous retrovirus (PERV). Taken together, we believe that the Biopsy Score enhancement of our islet isolation technique and our agarose-agarose macroencapsulation methodology bring us significantly closer to realizing clinical porcine islet xenotransplantation for the treatment of insulin-dependent diabetic patients.

A comprehensive microbiological safety approach for agarose encapsulated porcine islets intended for clinical trials.

The use of porcine islets to replace insulin‐producing islet β‐cells, destroyed during the diabetogenic disease process, presents distinct challenges if this option is to become a therapeutic reality for the treatment of type 1 diabetes. These challenges include a thorough evaluation of the microbiological safety of the islets. In this study, we describe a robust porcine islet‐screening program that provides a high level of confidence in the microbiological safety of porcine islets suitable for clinical trials.

Enhancement of In Vitro and In Vivo Function of Agarose Encapsulated Porcine Islets by Changes in the Islet Microenvironment

The transplantation of porcine islets of Langerhans to treat type 1 diabetes may provide a solution to the demand for insulin-producing cells. Porcine islets encapsulated in agarose–agarose macrobeads have been shown to function in nonimmunosuppressed xenogeneic models of both streptozotocin-induced and autoimmune type 1 diabetes. One advantage of agarose encapsulation is the ability to culture macrobeads for extended periods, permitting microbiological and functional assessment. Herein we describe optimization of the agarose matrix that results in improved islet function. Porcine islets (500 IEQs) from retired breeding sows were encapsulated in 1.5% SeaKem Gold (SG), 0.8% SG, or 0.8% Litex (Li) agarose, followed by an outer capsule of 5% SG agarose. Insulin production by the encapsulated islets exhibited an agarose-specific effect with 20% (0.8% SG) to 50% (0.8% Li) higher initial insulin production relative to 1.5% SG macrobeads. Insulin production was further increased by 40–50% from week 2 to week 12 in both agarose types at the 0.8% concentration, whereas islets encapsulated in 1.5% SG agarose increased insulin production by approximately 20%. Correspondingly, fewer macrobeads were required to restore normoglycemia in streptozotocin-induced diabetic female CD(SD) rats that received 0.8% Li (15 macrobeads) or 0.8% SG (17 macrobeads) as compared to 1.5% SG (19 macrobeads). Islet cell proliferation was also observed during the first 2 months postencapsulation, peaking at 4 weeks, where approximately 50% of islets contained proliferative cells, including β-cells, regardless of agarose type. These results illustrate the importance of optimizing the microenvironment of encapsulated islets to improve islet performance and advance the potential of islet xenotransplantation for the treatment of type 1 diabetes.

Hydrophilic Agarose Macrobead Cultures Select for Outgrowth of Carcinoma Cell Populations That Can Restrict Tumor Growth

Cancer cells and their associated tumors have long been considered to exhibit unregulated proliferation or growth. However, a substantial body of evidence indicates that tumor growth is subject to both positive and negative regulatory controls. Here, we describe a novel property of tumor growth regulation that is neither species nor tumor-type specific. This property, functionally a type of feedback control, is triggered by the encapsulation of neoplastic cells in a growth-restricting hydrogel composed of an agarose matrix with a second coating of agarose to form 6- to 8-mm diameter macrobeads. In a mouse cell model of renal adenocarcinoma (RENCA cells), this process resulted in selection for a stem cell-like subpopulation which together with at least one other cell subpopulation drove colony formation in the macrobeads. Cells in these colonies produced diffusible substances that markedly inhibited in vitro and in vivo proliferation of epithelial-derived tumor cells outside the macrobeads. RENCA cells in monolayer culture that were exposed to RENCA macrobead-conditioned media exhibited cell-cycle accumulation in S phase due to activation of a G(2)/M checkpoint. At least 10 proteins with known tumor suppression functions were identified by analysis of RENCA macrobead-conditioned media, the properties of which offer opportunities to further dissect the molecular basis for tumor growth control. More generally, macrobead culture may permit the isolation of cancer stem cells and other cells of the stem cell niche, perhaps providing strategies to define more effective biologically based clinical approaches to treat neoplastic disease.

No Evidence of Viral Transmission following Long-Term Implantation of Agarose Encapsulated Porcine Islets in Diabetic Dogs

We have previously described the use of a double coated agarose-agarose porcine islet macrobead for the treatment of type I diabetes mellitus. In the current study, the long-term viral safety of macrobead implantation into pancreatectomized diabetic dogs treated with pravastatin (n = 3) was assessed while 2 dogs served as nonimplanted controls. A more gradual return to preimplant insulin requirements occurred after a 2nd implant procedure (days 148, 189, and >652) when compared to a first macrobead implantation (days 9, 21, and 21) in all macrobead implanted animals. In all three implanted dogs, porcine C-peptide was detected in the blood for at least 10 days following the first implant and for at least 26 days following the second implant. C-peptide was also present in the peritoneal fluid of all three implanted dogs at 6 months after 2nd implant and in 2 of 3 dogs at necropsy. Prescreening results of islet macrobeads and culture media prior to transplantation were negative for 13 viruses. No evidence of PERV or other viral transmission was found throughout the study. This study demonstrates that the long-term (2.4 years) implantation of agarose-agarose encapsulated porcine islets is a safe procedure in a large animal model of type I diabetes mellitus.

Streptozotocin is responsible for the induction and progression of renal tumorigenesis in diabetic Wistar-Furth rats treated with insulin or transplanted with agarose encapsulated porcine islets.

Streptozotocin (STZ), a nitrosourea with DNA alkylating properties, has been widely used to induce hyperglycemia by specifically destroying the insulin-producing β-cells of the islets of Langerhans in experimental models of Type I diabetes. STZs known carcinogenic properties, however, raise concerns about its suitability for long-term studies. We conducted a formal study of STZs carcinogenic effects in long-term surviving diabetic Wistar-Furth rats. To determine if insulin therapy or islet transplantation exacerbated tumorigenesis, rats were randomly assigned to one of four experimental groups: normal animals with no treatment (Group 1, n=12); normal animals that underwent peritoneal implantation of porcine islets encapsulated in a double layer of agarose to form islet macrobeads (normal + islets; group 2, n=12); STZ treatment followed by daily exogenous insulin (STZ + insulin; group 3, n=18) and STZ treatment followed by the intraperitoneal implantation of porcine islet macrobeads (STZ + islets; group 4, n=14). At 215 days post-STZ induction, no renal proliferative lesions were observed in animals that did not receive STZ (group 1 and group 2) whereas adenoma incidences of 57% for group 3 and 34% for group 4 were observed. By terminal necropsy at day 351, the incidence and severity of renal proliferative lesions increased with tubular carcinoma observed in 67% of group 3 and 60% of group 4 animals. We conclude that the STZ-induced diabetic rat model is not suitable for long-term studies because of progressive renal tumorigenesis. Our experiments also demonstrate the safety and effectiveness of porcine islet macrobeads for the treatment of diabetes.

Pravastatin Improves Glucose Regulation and Biocompatibility of Agarose Encapsulated Porcine Islets following Transplantation into Pancreatectomized Dogs

The encapsulation of porcine islets is an attractive methodology for the treatment of Type I diabetes. In the current study, the use of pravastatin as a mild anti-inflammatory agent was investigated in pancreatectomized diabetic canines transplanted with porcine islets encapsulated in agarose-agarose macrobeads and given 80 mg/day of pravastatin (n = 3) while control animals did not receive pravastatin (n = 3). Control animals reached preimplant insulin requirements on days 18, 19, and 32. Pravastatin-treated animals reached preimplant insulin requirements on days 22, 27, and 50. Two animals from each group received a second macrobead implant: control animals remained insulin-free for 15 and 21 days (AUC = 3003 and 5078 mg/dL/24 hr days 1 to 15) and reached preimplant insulin requirements on days 62 and 131. Pravastatin treated animals remained insulin-free for 21 and 34 days (AUC = 1559 and 1903 mg/dL/24 hr days 1 to 15) and reached preimplant insulin requirements on days 38 and 192. Total incidence (83.3% versus 64.3%) and total severity (22.7 versus 18.3) of inflammation on tissue surfaces were higher in the control group at necropsy. These findings support pravastatin therapy in conjunction with the transplantation of encapsulated xenogeneic islets for the treatment of diabetes mellitus.

Improved Glucose Regulation on a Low Carbohydrate Diet in Diabetic Rats Transplanted With Macroencapsulated Porcine Islets

Islet xenografts from porcine donors can reverse diabetes in experimental animal models and may be an alternative to human islet transplantation. We have recently reported the ability of porcine islets encapsulated in a double layer of hydrophilic agarose to maintain in vitro functional ability for >6 months. Although β-cells are capable of adapting their secretory capacity in response to glucose levels, evidence has shown that prolonged hyperglycemia can compromise this ability. The aim of the present study was to determine the effects of diet manipulation on the long-term regulation of blood glucose levels, and the preservation of functional islet in the macrobeads. Twenty-one streptozotocin-induced diabetic Wistar-Furth male rats were randomly assigned to two diets containing 64% carbohydrate (CHO) or 20% CHO. Groups of five to six animals assigned to either diet were implanted with either empty (EM) or porcine islet-containing macrobeads (PIM) and followed for 333 days. Observations included general condition, body weight, blood glucose, and food and water intakes. Monthly blood samples were collected for insulin and C-peptide analysis. The 20% CHO diet significantly lowered blood glucose values when compared with those of the 64% CHO groups for both the empty (14.94 ± 0.41 vs. 16.26 ± 0.42 mmol/L, respectively, p < 0.001) and islet macrobead recipients (12.88 ± 0.39 vs. 15.57 ± 0.85 mmol/L, respectively, p < 0.001). The different diets, however, had no statistically significant effects on the preservation of islet mass in the macrobead. Serum porcine C-peptide was detected throughout the experiment in animals receiving porcine islet macrobeads, regardless of diet. Diabetic rats fed a low carbohydrate level diet and transplanted with porcine islet macrobeads had improved total tissue glucose disposal and improved clinical parameters associated with diabetes.