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Abstract

One challenge that must be overcome to allow transplantation of neonatal porcine islets (NPIs) to become a clinical reality is defining a reproducible and scalable protocol for the efficient preparation of therapeutic quantities of clinical grade NPIs. In our standard protocol, we routinely isolate NPIs from a maximum of four pancreases, requiring tissue culture in 16 Petri dishes (four per pancreas) in Ham's F10 and bovine serum albumin (BSA). We have now developed a scalable and technically simpler protocol that allows us to isolate NPIs from a minimum of 12 pancreases at a time by employing automated tissue chopping, collagenase digestion in a single vessel, and tissue culture/media changes in 75% fewer Petri dishes. For culture, BSA is replaced with human serum albumin and supplemented with Z-VAD-FMK general caspase inhibitor and a protease inhibitor cocktail. The caspase inhibitor was added to the media for only the first 90 min of culture. NPIs isolated using the scalable protocol had significantly more cellular insulin recovered (56.9 ± 1.4 μg) when compared to the standard protocol (15.0 ± 0.5 μg; p < 0.05). Compared to our standard protocol, recovery of β-cells (6.0 × 10⁶ ± 0.2 vs. 10.0 × 10⁶ ± 0.4; p < 0.05) and islet equivalents (35,135 ± 186 vs. 41,810 ± 226; p < 0.05) was significantly higher using the scalable protocol. During a static glucose stimulation assay, the SI of islets isolated by the standard protocol were significantly lower than the scale-up protocol (4.3 ± 0.2 vs. 5.5 ± 0.1; p < 0.05). Mice transplanted with NPIs using the scalable protocol had significantly lower blood glucose levels than the mice that receiving NPIs from the standard protocol (p < 0.01) and responded significantly better to a glucose tolerance test. Based on the above findings, this improved simpler scalable protocol is a significantly more efficient means for preparing therapeutic quantities of clinical grade NPIs.

Keywords

Neonatal porcine islets (NPIs)Islet transplantation Islet isolation

Introduction

An attractive alternative to daily insulin injections is to transplant insulin-producing tissue to achieve a more physiological means for restoring glucose homeostasis, thereby potentially reversing the metabolic and neurovascular complications of diabetes. Seven patients transplanted in 2000 by the Islet Transplant Group in Edmonton attained insulin independence by receiving freshly isolated islets from multiple donors and steroid-free antirejection therapy—a procedure known as the Edmonton Protocol (28,29,32). Modifications of this protocol by other groups have also resulted in similar successes (16,21,30,33,34,37). To date, in Edmonton, approximately 300 patients have been transplanted; this is less than anticipated, in large part because of the limited supply of cadaveric pancreases (32). To allow islet transplantation to become a more widespread form of therapy for more patients with type 1 diabetes, an unlimited source of islets must be identified.

There is a strong rationale to pursue the use of porcine donors as an unlimited source for clinical islet xenotransplantation. The principle challenges that must be met to make xenotransplantation of porcine islets a clinical reality include defining a reproducible strategy for the efficient preparation of large numbers of clinical grade islets, understanding and minimizing the risk of transmission of porcine pathogens, and overcoming the patient rejection barrier with clinically applicable immunosuppression and ultimately tolerance induction strategies. While other groups report protocols for the isolation of adult (9) or juvenile porcine islets (22,23), our group reported a simple, inexpensive, and reproducible method to isolate large numbers of neonatal porcine islets (NPIs) (20). These islets are comprised of differentiated endocrine and endocrine precursor cells that both in vitro and in vivo have the potential for proliferation and differentiation and have been shown to reverse hyperglycemia in immunodeficient mice (20), allogeneic outbred pigs (19), and moreover in nonhuman primates (7,35). Furthermore, NPIs are appealing because of their resistance to hypoxia (11), human proinflammatory cytokines (14), hyperglycemia (18), and toxicity of islet amyloid deposition (26), as well as their inherent ability to differentiate and proliferate (20) and achieve transplant tolerance induction in diabetic mice (1). Taken together, these observations clearly indicate that neonatal porcine islets are an ideal source of tissue for clinical islet xenotransplantation.

One principal challenge that must be met to make transplantation of neonatal porcine islets a clinical reality is defining a reproducible and scalable strategy for the efficient preparation of therapeutic quantities of clinical grade neonatal porcine islets. In our standard laboratory protocol (20), we routinely isolated islets from only four neonatal porcine pancreases, but the protocol is very laborious and at a high risk for potential contamination. A more scalable, technically simpler and clinically acceptable protocol was developed in this study. We modified the isolation protocol to accommodate larger numbers of pancreases and simplified the culture procedure to reduce the amount of culture Petri dishes.

The protocol developed by this study will facilitate and expedite clinical trials of neonatal porcine islets, thereby allowing the treatment of many more patients with type 1 diabetes.

Materials and Methods

Neonatal Porcine Islet Preparation

Donor pancreases were obtained from 1- to 3-day-old Duroc neonatal piglets (male or female) from the University of Alberta Swine Research Centre (1.5–2.0 kg body weight), and the islets were isolated and cultured for 5–7 days as described previously (20) (Fig. 1A). For our previously published standard protocol (Fig. 1A), the retrieved pancreases (four per isolation) were cut into 1- to 3-mm tissue fragments in separate 50-ml conical tubes using scissors, digested with collagenase (type XI, 2.5 mg/ml; Sigma-Aldrich, Oakville, Ontario, Canada), filtered through a 500-μm nylon screen, washed in Hank's balanced salt solution (HBSS; Gibco, Burlington, Ontario, Canada) supplemented with 0.25% bovine serum albumin (BSA; fraction V; Sigma-Aldrich), 10 mM HEPES (ICN Biomedicals, Inc., Costa Mesa, CA, USA), 100 U/ml penicillin, and 0.1 mg/ml streptomycin (Lonza, Inc., Walkersville, MD, USA). The digested tissue was then cultured in four nontissue culture-treated 150-mm-diameter Petri dishes per pancreas (Fig. 1A; 16 Petri dishes per isolation) in 35 ml of Ham's F10-BSA tissue culture media (Sigma-Aldrich) supplemented with 14.3 mM sodium bicarbonate (Thermo Fisher Scientific, Ottawa, Ontario, Canada), 10 mM D-glucose (Electron Microscopy Sciences, Hatfield, PA, USA), 2 mM L-glutamine (Sigma-Aldrich), 0.25% BSA (fraction V; Sigma-Aldrich), 50 μM isobutylmethylxanthine (IBMX; Sigma-Aldrich), 10 mM nicotinamide (Sigma-Aldrich), 1.6 mM calcium chloride dihydrate (Sigma-Aldrich), 100 U/ml penicillin, and 0.1 mg/ml streptomycin (Lonza). The islets in this standard four Petri dish protocol were cultured at 37°C for 5–7 days, with the medium changed the first, third, and fifth days after isolation. A control group included pancreases processed as outlined above, but the digest from each pancreas was cultured in one Petri dish (standard one Petri dish protocol) as opposed to four Petri dishes per pancreas (standard four Petri dish protocol).

Figure 1.

Protocols for isolating neonatal porcine islets. Schematic diagram of the isolation of neonatal porcine islets using our standard protocol (A) and cultured in Ham's-BSA (four Petri dishes/pancreas) or a scalable protocol and cultured in Ham's-HSA-CI-PI (one Petri dish/pancreas) (B).

For the experimental scalable protocol (Fig. 1B), 12 pancreases per isolation were surgically removed and then cut into 1- to 2-mm³ fragments in 160 ml HBSS without BSA using an automated tissue chopper (Retsch Grindomix GM 200; Verder Scientific, Haan, Germany) at 2,000 rpm in two 5-s bursts (Fig. 1B). The tissue from all 12 pancreases was then digested with collagenase in a single 225-ml conical tube, then filtered through a 500-μm nylon mesh. The combined tissue was cultured in one 150-mm Petri dish per pancreas (12 dishes for 12 pancreases) in 35 ml Ham's F10 as described above but supplemented with 0.5% human serum albumin (HSA) replacing the 0.25% BSA and a protease inhibitor (PI) cocktail (1:500; Sigma-Aldrich). For the first 90 min of culture, the media also contained 11.1 μM Z-VAD-FMK general caspase inhibitor (CI) (R&D Systems, Minneapolis, MN, USA), then the islets were washed twice in HBSS and cultured in the same media but with no CI. The islets were cultured in Ham's F10-HSA-PI at 37°C for 5–7 days, with the medium changed the first, third, and fifth days after isolation. NPIs cultured according to our standard protocol with four plates per pancreas were at a density of less than 1 IEQ/mm², while NPIs cultured in one plate per pancreas were at a density of approximately 2 IEQ/mm².

In Vitro Assessment of Islets

Following tissue culture, recovery of the NPI preparations was determined on the basis of cellular insulin and DNA content as well as IEQs (15). All measurements were assessed from duplicate aliquots of the NPI suspensions. Cellular insulin content was measured after extraction in 2 mM acetic acid containing 0.25% BSA (15). Samples were sonicated in acetic acid, centrifuged at 800 × g for 15 min, and then supernatants were collected and stored at −20°C until assayed for insulin content by ELISA (Roche Diagnostics, Laval, Quebec, Canada). For DNA content, representative aliquots were washed in citrate buffer [150 mmol/L NaCl (Sigma-Aldrich), 15 mmol/L citrate (Sigma-Aldrich), 3 mmol/L EDTA (Sigma-Aldrich), pH 7.4] and stored as cell pellets at −20°C before being assayed by Picogreen (Molecular Probes, Inc., Eugene, OR, USA), a fluorescent nucleic acid stain for quantification of double-stranded DNA (15). Aliquots from each preparation were also counted and sized to determine the recovery of IEQs from each NPI preparation (15).

For assessment of in vitro viability, a static incubation assay (20) was used to determine glucose stimulated insulin secretion of NPIs prepared using each protocol. Fifty to 100 representative NPIs from each condition were incubated in duplicate at 37°C for 2 h in 1.5 ml RPMI (Sigma-Aldrich) supplemented with 2.0 mM L-glutamine (Sigma-Aldrich), 0.5% w/v BSA, and either 2.8 mM (low) or 20.0 mM (high) glucose (11). Culture supernatant was collected, stored at −20°C, and measured for insulin at a later time by a porcine insulin immunoassay (Meso Scale Discovery, Gaithersburg, MD, USA). Stimulation indices were calculated by dividing the amount of insulin released at 20.0 mM glucose by that released at 2.8 mM glucose.

Immunohistochemistry was used to determine the cellular composition of the NPIs. The avidin-biotin complex (ABC) method was used with peroxidase and 3,3′-diaminobenzidine (DAB) (Sigma-Aldrich) as the chromagen. NPIs were dissociated into single cells mechanically with a siliconized Pasteur pipette in a solution of 0.05% trypsin, 0.5 mM EDTA, and phosphate buffered saline (PBS) (20). The single cells were then fixed in formaldehyde (Sigma-Aldrich) on glass slides and stained to determine the proportion of insulin, glucagon, and cytokeratin-7 (CK-7) positive ductal cells. Primary antibodies (Dako Corp., Carpinteria, CA, USA) included guinea pig anti-porcine insulin (1:1,000), rabbit anti-glucagon (1:100), and mouse anti-human CK-7 antibody (1:200); biotinylated secondary antibodies and the ABC-enzyme complexes were purchased from Vector Laboratories (Burlingame, CA, USA). Primary antibodies were incubated for 30 min at room temperature, while secondary antibodies were applied for 20 min.

Using the total DNA content and the known quantity of 7.1 pg DNA per cell (15), combined with the proportion of stained cells from the immunohistochemistry, we were able to calculate the number of positively stained cells by the following formula:

\begin{array}{l} \frac{Total DNA content}{7.1 pg DNA/cell} \times \frac{% positive cells}{100} \\ = number of positive stained cells \end{array}

Essentially, this methodology allows us to accurately calculate the number of insulin-, glucagon-, and CK-7-positive cells.

Transplantation and Metabolic Follow-up

Following culture, NPIs from either the clinically applicable protocol (n = 20) or standard laboratory protocol (n = 20) were transplanted under the left kidney capsule of 8-week-old halothane-anesthetized male (Jackson Laboratory, Bar Harbour, ME, USA) diabetic B6.Rag^-/-mice (15). All mice were fed with standard laboratory food and cared for according to the guidelines established by the University of Alberta Animal Care and Use Committee and the Canadian Council on Animal Care Committee, and our protocol was approved by these agencies. Mice were rendered diabetic by intravenous injection of 185 mg/kg body weight streptozotocin (Sigma-Aldrich) 4–5 days before transplantation. All recipients entering this study exhibited blood glucose levels above 20 mmol/L. Blood samples were obtained from the tail vein for glucose assay (OneTouch glucose meter; LifeScan Canada Ltd., Burnaby, Canada). Animals were maintained in climatized rooms with free access to sterilized tap water and pelleted food. Aliquots consisting of 2,000 NPI IEQs were aspirated into polyethylene tubing (PE-50), pelleted by centrifugation, and gently placed under the kidney capsule with the aid of a micromanipulator syringe. Once the tubing was removed, the capsulotomy was cauterized with a disposable high-temperature cautery pen (Bovie Medical Corporation, Clearwater, FL, USA).

Transplanted mice were monitored for blood glucose levels once a week between 8:00 and 11:00 a.m. The graft was deemed a success when the blood glucose level was ≤8.4 mmol/L. At posttransplantation week 12, an oral glucose tolerance test (OGTT) was performed on all NPI recipients with normalized basal glycemia (n = 20 from each of the standard laboratory and clinically applicable protocols). After a 2-h fast, D-glucose (3 mg/g body weight) was administered as a 50% solution intragastrically into nonanesthetized mice. Blood samples were obtained from the tail vein at 0, 30, 60, 90, and 120 min. Age-matched diabetic and normal mice were included as controls. Following the OGTT, survival nephrectomies were performed on the graft-bearing kidneys of transplanted mice, and the mice were monitored until hyperglycemia returned to ensure the reversal of diabetes was attributed to the graft and not β-cell regeneration.

Statistical Analysis

All statistics were performed using the one-way analysis of variance (ANOVA) with the Bonferroni correction for multiple groups or the Student's t-test for two groups; the level of significance was considered to be p ≤ 0.05. All statistical analyses were performed with STATA 11 (StataCorp LP, College Station, TX, USA). Results are presented as mean ± standard error of the mean (SEM).

Results

NPI Preparation

NPIs were isolated using our previously published (20) standard protocol and cultured in four Petri dishes per pancreas in Ham's F10-BSA (Fig. 1A; n = 16 isolations of four pancreases per isolation) or a modified scalable protocol and one Petri dish per pancreas in Ham's F10-HSA-CI-PI with BSA replaced with HSA and the addition of CIs and PIs (Fig. 1B; n = 4 isolations of 12 pancreases per isolation). As a control, NPIs isolated according to the standard laboratory protocol were also cultured in Ham's F10-BSA at a density of one Petri dish per pancreas (n = 6 isolations of four pancreases per isolation). Using the scalable protocol and Ham's F10-HSA-CI-PI, significantly more cellular insulin content was recovered (56.9 ± 1.4 μg) when compared to the standard laboratory protocol and culturing in either four Petri dishes (15 ± 0.5 μg) or one Petri dish per pancreas (9.0 ± 0.3 μg) (p < 0.05) (Table 1). A significantly higher proportion (p < 0.05) of insulin-positive β-cells were also recovered using the scalable protocol (31 ± 1.5%) compared to the standard protocol with either four Petri dishes (21 ± 0.2%) or one Petri dish (24 ± 0.8%) per pancreas (Table 1). Similarly, using the scalable protocol and Ham's F10-HSA-CI-PI, the total number of β-cells (10 ± 0.4 × 10⁶) and IEQs (41,810 ± 226) recovered per pancreas were also significantly higher (p < 0.05) compared to the standard protocol and Ham's F10-BSA when cultured using either four (6 ± 0.2 × 10⁶; 35,135 ± 186, respectively) or one (7 ± 0.2 × 10⁶; 38,649 ± 254, respectively) Petri dishes (Table 1). In contrast, there were no differences between numbers of glucagon positive α-cells recovered per pancreas between the three protocols (Table 1). However, there were significantly fewer (p < 0.05) CK-7-positive cells recovered per pancreas using the scalable protocol and Ham's F10-HSA-CI-PI (4 ± 0.1 × 10⁶) when compared to standard protocol and Ham's F10-BSA in either four (8 ± 0.2 × 10⁶) or one Petri dish (8 ± 0.8 × 10⁶).

Table 1.

Comparison of Neonatal Porcine Islets Isolated and Cultured in Our Standard Laboratory Culture Protocol Compared to a Simplified Scaled-Up Protocol

	Content/Pancreas (μg)
Condition	Insulin	DNA	Cell Composition (% of Total) (β/α/CK-7)	Cell No./Pancreas (10⁶) (β/α/CK-7/Total)	IEQ/Pancreas
A	15.0 ± 0.5^*	208.0 ± 6.8	21 ± 0.2/17 ± 0.2/25 ± 0.5^	6 ± 0.2/5 ± 0.2/8 ± 0.2/31 ± 1.0	35,135 ± 186^*
B	9.0 ± 0.3^*	227.0 ± 10.6	24 ± 0.8/21 ± 0.4/25 ± 0.8^	7 ± 0.2/6 ± 0.4/8 ± 0.8/30 ± 1.6	38,649 ± 254^*
C	56.9 ± 1.4	238.0 ± 3.0	31 ± 1.5/17 ± 1.5/13 ± 0.5	10 ± 0.4/6 ± 0.5/4 ± 0.1/34 ± 0.5	41,810 ± 226

Data are means ± SEM. Islets were isolated and cultured using condition A: our standard protocol in four Petri dishes/pancreas in Ham's F10 BSA (n = 16 isolations of four pancreases per isolation; n = 64 pigs); condition B: our standard protocol in one Petri dish/pancreas in Ham's F10 BSA (n = 6 isolations of four pancreases per isolation; n = 24 pigs); and condition C: scaled-up protocol using one Petri dish per pancreas in modified Ham's F10 with BSA replaced with HSA and addition of caspase and protease inhibitors (n = 4 isolations of 12 pancreases per isolation; n = 48 pigs).

p < 0.05 versus scaled-up protocol.

NPI Insulin Secretory Responsiveness In Vitro

During a static glucose stimulated insulin secretion assay, significantly more insulin was secreted at high glucose (20.0 mM) compared to low glucose (2.8 mM) in all three experimental conditions (Table 2; significance not shown). Significantly less insulin was secreted at low glucose by islets isolated using the scalable protocol (1.5 ± 0.04% of total cellular insulin content) compared to islets isolated according to the original protocol following culture in one Petri dish per pancreas (1.8 ± 0.1%; p < 0.05) but not four Petri dishes per pancreas (1.9 ± 0.1%) (Table 2; indicated by *). There were no differences between the amount of insulin released at high glucose by islets isolated using the standard protocol with culture in four (8.1 ± 0.5%) or one (7.9 ± 0.3%) Petri dishes per pancreas compared to each other or compared to the scalable protocol (8.2 ± 0.1%). When calculating the stimulation indices, there were no significant differences between stimulation indices of NPIs cultured according to the standard protocol in either four (SI: 4.3 ± 0.2) or one (SI: 4.4 ± 0.2) Petri dish per pancreas. However, when compared to both standard protocol conditions, those NPIs isolated and cultured using the scalable protocol exhibited a significantly higher (p < 0.05) stimulation index (5.5 ± 0.1) (Table 2).

Table 2.

Glucose-Stimulated Insulin Secretion of Islets Isolated and Cultured in Our Standard Laboratory Culture Protocol Compared to a Simplified Scaled-Up Protocol

	% Cellular Insulin Content
Condition	2.8 mM	20 mM	SI
A	1.9 ± 0.1^*	8.1 ± 0.5	4.3 ± 0.2^*
B	1.8 ± 0.1^*	7.9 ± 0.3	4.4 ± 0.2^*
C	1.5 ± 0.04	8.2 ± 0.05	5.5 ± 0.1

p < 0.05 versus scaled-up protocol.

Transplantation of NPIs in Diabetic Mice

All mice transplanted with NPIs from either the standard protocol (n = 20 mice) or the scalable protocol (n = 20) achieved normoglycemia (Fig. 2A). However, 100% of the mice transplanted with NPIs prepared from the scalable protocol became euglycemic within 8 weeks posttransplantation, while the mice implanted with NPIs using the standard protocol did not normalize until week 10 posttransplant. From week 2 until survival nephrectomies at week 12 posttransplant, mice grafted with islets from the scalable protocol had significantly lower glycemias than mice transplanted with NPIs prepared using the standard protocol (p < 0.001). In both groups, removal of the graft-bearing kidneys resulted in a rapid return to the diabetic state, indicating that the NPI grafts were responsible for normoglycemia.

Figure 2.

Blood glucose values of mice transplanted with neonatal porcine islets. (A) Weekly blood glucose values of mice transplanted with neonatal porcine islets prepared from either our standard protocol (filled square; n = 20) or clinically applicable protocol (filled diamond; n = 20). At week 12 posttransplant, all mice underwent a nephrectomy of the graft-bearing kidney (arrow) to ensure a return to normoglycemia. *p < 0.01. (B) Blood glucose values during oral glucose tolerance tests in mice transplanted with neonatal porcine islets prepared from either our standard protocol (filled square; n = 20) or clinically applicable protocol (filled diamond; n = 20). Normal mice (filled circle; n = 5) and diabetic mice (open circle; n = 5) were also included as controls. *p < 0.01.

OGTTs performed at 12 weeks posttransplant demonstrated that mice transplanted with NPIs prepared using the scalable protocol (filled diamond; n = 20) had significantly lower blood glucose levels (p < 0.01) at every time point during the OGTTs compared to mice implanted with NPIs using the standard protocol (filled square; n = 20), normal mice (filled circle; n = 5), and diabetic mice (open circle; n = 5) (Fig. 2B). In all groups except the normal controls, the glycemia at 120 min after the bolus of glucose was not significantly different from the values at 0 min (p > 0.05; for normal controls, p < 0.01).

Discussion

One challenge that must be overcome to allow transplantation of NPIs to be a clinical reality is defining a reproducible and scalable protocol for the efficient preparation of therapeutic quantities of clinical grade NPIs. In our previously published standard protocol (15), we isolated NPIs from a maximum of four neonatal porcine pancreases that required tissue culture in 16 Petri dishes (four per pancreas) (Fig. 1A). Furthermore, based on our previously published nonhuman primate studies, at least 12 neonatal porcine pancreases will be required to reverse diabetes in human patients (8,12).

We have now developed a more scalable and technically simpler protocol that allows us to isolate NPIs from a minimum of 12 pancreases at a time by employing automated tissue chopping, collagenase digestion in a single vessel, and tissue culture/media changes in 75% fewer Petri dishes (Fig. 1B). In this new culture media, BSA was replaced with the clinically acceptable HSA and the CIs and PIs were added, since tissue from one pancreas was cultured in one not four Petri dishes and was therefore significantly more concentrated. While the scalable protocol is faster, less laborious, more efficient, and has lower probability of contamination, it also significantly increased the recovery of total cellular insulin content, absolute number of β-cells, as well as IEQs recovered per pancreas (Table 1). In addition, the scalable protocol also significantly improves the in vitro glucose-stimulated insulin secretory response of the NPIs as indicated by higher stimulation indices after a static incubation (Table 2).

Moreover, the scalable protocol significantly ameliorates the restoration of glycemia and the glucose tolerance of transplanted mice compared to the standard protocol, normal, and diabetic mice. The differences between the OGTTs of mice transplanted with NPIs from the scalable protocol and normal mice are as expected because the physiological blood glucose levels for porcine islets are lower than those for mouse islets (20). The superior glucose response of NPIs isolated and cultured according to the scalable protocol compared to the standard protocol is likely due to the increased proportion of β-cells and insulin secretory capacity of NPIs isolated using the scalable protocol over the standard protocol. This enhanced function of NPIs isolated with the scalable protocol is potentially due to decreased β-cell apoptosis as a result of the short-term addition of the general CI (4 –6,8,24,25). Pretreatment of human (4) or adult porcine (5) islets with caspase 3 inhibitors for 24–48 h by other groups show significant decreases in islet viability and function both in vivo and in vitro. The positive effect of 90-min pretreatment with Z-VAD-FMK in NPIs could be attributed to the fact that it is a general CI instead of only caspase 3 and exposure time was much shorter (25), as well as the inherent resistance of NPIs to apoptosis after hypoxia (11). Serine PIs have been demonstrated to have a protective effect on the membrane integrity of rat islets upon exposure to collagenase (36); this is likely another contributing factor to the improved viability and function of the islets.

It is likely that the β-cells are especially sensitive to apoptosis, resulting in the increased β-cell content in the NPIs from the scalable protocol without changing the α-cell content due to the CI (25). The significantly lower proportion of CK-7-positive cells could also contribute to the increased number of β-cells; a 2004 study first showed a clonal population of pancreatic precursor cells rising from the ductal cell population, possibly linked through a neural stem cell lineage (10,31). Subsequently, the ability of human pancreatic duct cells to differentiate into β-cells has been demonstrated by multiple groups (2,3,15,17,38). Thus, it is possible that our scalable protocol encourages differentiation of these precursors into β-cells; however, this remains to be investigated.

Based on the above findings, this improved scalable protocol could be utilized to manufacture a clinical neonatal porcine islet product. This protocol could now be tested using clinically acceptable reagents such as replacing Ham's F10 tissue culture media and collagenase with good manufacturing practice (GMP) certified products (27). GMP is a required regulatory approach recognized worldwide for ensuring the quality control of manufactured pharmaceuticals, blood products, and medical devices. This designation guarantees that the cells and tissues will be consistently manufactured in strictly defined physical environments using high-quality systems and closely controlled procedures. The pancreatic chopper apparatus used in this study can be validated to comply with these GMP standards or replaced with an apparatus that has already received such certification (13). In addition to clinically approved reagents and protocols, a GMP laboratory is required for producing live therapeutic cells and tissues for cell therapy treatments. FDA and Health Canada requires GMP certification for clinical grade cells and tissues produced for use in clinical trials. The protocol described in this study can thus be further adapted to comply with GMP regulations.

Footnotes

Acknowledgments

This study is supported by the Canadian Institutes of Health Research (Grant #MOP 119500), Juvenile Diabetes Research Foundation (#17-2013-286), and Canadian Stem Cell Network. C.E. is a recipient of an Alberta Innovates Health Solutions Studentship. We would like to thank the University of Alberta Swine Research Centre for the neonatal piglets and Deb Dixon for assistance with isolating the NPIs. The funders had no role in study design, data collection and analysis, decision to publish, or publication of manuscript. Author contributions: Cara Ellis—drafting article, data collection, data analysis/interpretation; James Lyon—conducted experiments, data collection; Greg Korbutt—concept/design, data analysis/interpretation, critical revision of article, securing funding. The authors declare no conflicts of interest.

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Optimization and Scale-up Isolation and Culture of Neonatal Porcine Islets: Potential for Clinical Application

Abstract

Keywords

Introduction

Materials and Methods

Neonatal Porcine Islet Preparation

In Vitro Assessment of Islets

Transplantation and Metabolic Follow-up

Statistical Analysis

Results

NPI Preparation

NPI Insulin Secretory Responsiveness In Vitro

Transplantation of NPIs in Diabetic Mice

Discussion

Footnotes

Acknowledgments

References