A method for the massive separation of highly purified, adult porcine islets of langerhans

A Method for the Massive Separation of Highly Purified, Adult Porcine Islets of Langerhans Riccardo Calafiore, Albert0

Filippo Calcinaro,

Giuseppe Basta, Massimo

Falorni, Maurizio Piermattei,

Pietropaolo,

and Paolo Brunetti

A method for the massive and reproducible isolation of highly purified, adult porcine islets of Langerhans is described. The successful combination of donor animal-strain selection with original procedures for pancreas retrieval and enzymatic digestion permitted us to separate uniquely massive concentrations of pure porcine islets with no need for mechanical disruption of the pancreatic tissue. Following our procedure, porcine islets, which fully retain viability and function, can be harvested easily and rapidly. Xenotransplantation of such islets, immunoprotected within algin/polyaminoacidic microcapsules, was associated with complete reversal of hyperglycemia in rodents with either spontaneous or streptozotocininduced diabetes mellitus. 0 1930 by W.B. Saunders Company.

T

HE RESTRICTED availability of cadaveric human donor pancreata continues to hamper progress of islet cell transplantation into clinical trials. An attractive alternative would be to employ non-human pancreatic tissue as a resource for donor islets. We have developed a method for the large-scale isolation of highly purified islets of Langerhans from the pancreas of adult pigs. These islets could be ideal candidates as donor pancreatic tissue, provided that their immunologic patterns were altered, because of both the molecular affinity between pork and human insulin and the large availability of pigs. In comparison with procedures previously described,’ our method provides incomparably higher and uniquely massive concentrations of pure porcine islets. We report that such islets have been proven to retain full viability as well as functional competence, either in vitro or in vivo, after xenograft in

diabetic rodents. MATERIALS AND METHODS

Organ Procurement A local slaughterhouse provided us with female adult pigs (breeding strain: Large White), 8 months old, weighing an average 100 kg., that received no treatment prior to pancreas retrieval. The porcine pancreas has a duodenal and splenic lobe. Since the latter has been shown to contain larger concentrations of islets, we processed only this portion of the organ. The pancreas was retrieved, under fairly sterile conditions, immediately after the animals were shot through the head but prior to cardiac arrest, with care being taken to avoid significant bleeding. These maneuvers were performed to reduce the overall ischemia time and to protect islet viability. Once isolated the pancreas was placed into a sterile bag containing Hank’s balanced salt solution (HBSS) (Flow, Rickmansworth, UK), supplemented with 10% bovine serum (Flow) and antibiotics (Flow) at 4OC. The organ was immediately transported to the laboratory and handled within 45 minutes of retrieval. Islet Isolation A multienzymatic solution (Table 1) whose final volume, in milliliters, was equal to twice the pancreas weight, in grams, was prepared. After removing all mesenteric leaflets and the tail fibrous cap, the organ was considered clean and cannulated, via the main duct, with an indwelling, suture-secured, 16-gauge polyethylene catheter (Abbocath T-16, Abbott Laboratories LTD. Queensborough, UK). A volume of the enzymatic solution in milliliters, equal to the pancreas Metabolism, Vol39,

No 2 (February). 1990: pp 175-l 8 1

weight in grams, was forced through the cannulated duct, throughout overall four to five minutes, by a 60-mL plastic syringe (Becton Dickinson, Dun Laoghaire Co LTD, Dublin, Ireland), which resulted in visible distention of the organ. The distended pancreas was then immersed in an apparatus, driven by a peristaltic pump (Minipuls 2, Gilson Medical Electronics, Inc, Middleton, WI) that replaced the syringe, in a bath at 37% containing the balance of the enzymatic solution and continuously perfused at a Row rate of 7 mL/min (Fig 1). Pressures generated in the pancreas were 15 to 20 and 3 bars, during manual distention and perfusion, respectively. The solution seeped through the organ and exited into the aqueous bath where it was, once again, drawn up by the pump and recirculated through the pancreas for overall 30 minutes. The organ and aqueous bath were thereafter shaken at 110 cycles/min at 37OC, for an additional 10 minutes, when the pancreatic tissue came finely apart. Digestion time was established after processing 70 pancreata that were retrieved from Large White pigs of comparable age, sex, and body weight. The preparation was, at this point, chilled with 200 mL of cold HBSS supplemented with 10% heat-inactivated newborn calf serum (Flow). Occasional undigested tissue chunks were discarded, since they were not proven to affect the final yield in viable islet cells. This process disengaged massive concentrations of islets, making them float away from the remainder of the pancreatic tissue and collect in a wide portion of the supernatant. Therefore a pancreatic tissue suspension that was already enriched in free islets was easily and rapidly harvested simply by skimming it from surface layers of the aqueous bath, under gentle beaker shaking, by a stainless, 14-gauge cannula (American Scientific Products, Miami, FL) mounted on a 60-mL plastic syringe (Fig 2). This process continued, by diluting tissue suspension with cold HBSS, several times until islets were shown in the supernatant. Hence following this method there was no need for mechanical disruption of the digest through choppers, macerators, or other devices, since appropriate enzymatic digestion alone was associated with massive liberation of islets. After several washes in HBSS at 450 g at 4OC for 5 minutes each, islet tissue, whose packed cell volume never exceeded 20 mL. was then resus-

From the Istituto di Patologia Speciale Medica e Metodologia Clinica, University of Perugia, School of Medicine, 06100 Perugia and Consorzio interuniversitario per il Trapianto d’Organo (DPR 31/l O/1 988). Italy. Supported by Grant No. 8700201.56, given by the Italian National Councilfor Research (CNR). Address reprint requests to Riccardo CalaJiore. MD, Istituto di Patologia Speciale Medica, Via E. Dal Pozzo, 06100 Perugia, Italy. o 1990 by W.B. Saunders Company. 0026-0495/90/3902-0011%3.00/O

175

176

CALAFIORE ET AL

Table 1. Multienzymatic Solution for the Digestion of Porcine Pancreas Reagent

SpecificActivity

Source

450 Ujmg solid

Sigma

2 mg/mL

45 U/mg solid

Sigma

0.5 mg/mL

Sigma

0.3 mg/mL

Sigma

1 mg/mL

Concentration

Collagenasetype V Elastase Deoxyribonu-

Islet Culture Tissue aliquots, comprised of 100,000 islets, were placed in 100 x 15 mm Petri dishes (Becton Dickinson Labware, Lincoln Park, NJ) containing 10 mL of RPM1 1640 supplemented with heatinactivated 10% fetal calf serum (Flow) and antibiotics and thereafter incubated at 37OCin humid atmosphere of 95% air/CO,. Culture media were replaced every 48 hours.

600 Kunitz U/

cleasa

mg protein Calcium chloride (anydrous)

pended at room temperature in a solution of Ficoll400 DL (Sigma Chemical Co, St. Louis, MO) in HBSS to a final density of 1,110. Discontinous gradients were prepared, with slight modifications of procedures previously described? by layering 15 mL of Ficoll/ HBSS, at densities of 1,090, 1,080 and 1,040 each on 80-mL 1,100 density tissue/Ficoll aliquots in round-bottom 150-mL glass bottles. The gradients were centrifuged at 400 g for 35 minutes at 4°C. Massive concentrations of pure islets were collected from the top of the 1,040 Ficoll layer, whereas no islets but only nonendocrine pancreatic cell components were found in the other layers and in the tissue pellet. The final, packed, islet-cell volume, assessed after harvest and wash in HBSS, at 450 g and 4OCranged from 0.4 to 0.6 ml, with the estimated purity of the islets, as assessed by light microscopy (see below), overcoming 95%. Assessment of Islet Yield The final islet yield was assessed under inverted phase microscope (Jenoptik Jena GmbH, Jena, DDR) equipped with a grid in the eyepiece, by counting random aliquots of the islet suspension. Prior to counting, islets were incubated with an alcoholic solution of 0.03% Diphenylthiocarbazone (DTZ, Sigma Chemical Co), which stains specifically in red insulin-containing tissue,3 thus facilitating quantitative and qualitative assessment of the final islet preparation. Bach islet count was performed as follows: After diluting the final islet suspension with 15 mL RPM1 1640 (Flow) under gentle agitation, a 0.2-mL random aliquot was transferred to a 50-mm Petri dish for count. Average 10,000 islets were examined each time. Only islets whose diameter resulted higher than 60 pm (see below) were quoted for the assessment of the final islet yield. Insulin content was measured by acid-ethanol extraction in aliquots of the final islet preparation and expressed as insulin recovery/g of pancreatic tissue.

Morphological Studies Isolated islets were examined either at the end of the isolation or after 48- to 72-hour incubation periods. Islet aliquots and specimens of the native pancreas were fixed in 10% buffered formaldehyde, dehydrated with ethanol, and finally embedded in paraffin. Microtome sections, 5 p thick, were mounted on glass slides and stained with aldheyde fucsin. Additional islet aliquots were examined by electron microscopy after processing preparations in agreement with procedures previously described.4 Viability of fresh islets was also assessed by staining with ethidium bromide and fluorescein diacetate’ under fluorescence microscope (Leitz Orthomat IV, Ernst Leitz Wetzlar GmbH, Wetzlar, West Germany). Size distribution of either isolated or native pancreatic islets was assessed by morphometric evaluations of sections stained with aldheyde fucsin, with a light microscope equipped with a 240-mm scaled screen (Reichert Univar, Vienna, Austria). In Vitro Studies Porcine islet insulin-secretory function was assessed in either static incubation or perifusion studies. Batches of 25 islets were either incubated for 2 hours at 37OCwith HBSS containing glucose at different concentrations (5.5 mmol/L to 16.5 mmol/L), with or without 0.1 mmol/L 3-isobutyl-1-methylxanthine (IBMX) and 10 mmol/L arginine or continuously perifused for overall 2 hours with the same secretagogues according to methods previously described.6 At the end of each study media were collected for insulin assay. In Vivo Studies After 24 hours culture, islets were enveloped within algin polyaminoacidic microcapsules according to methods previously described.6 Polymer/islet mixture was adjusted so as to make each capsule contain an average of four to six islets. Microencapsulated porcine islets, which were associated with full retention of physiologic insulin-secretory kinetics in vitro were xenoengrafted, after an

peristaltic pump \

C

C

enzymatic

solution

-

pancreas

bath Figl. Schematic representations of the apparatus for enzymatic digestion of the porcine pancreas.

177

MASSIVE SEPARATION OF PORCINE ISLETS OF LANGERHANS

r====l -ADIPOSE

TISSUE

FREE ISLETS Fig 2. Schematic representation of porcine pancreatic digest at the end of the perfusion.

PELLET

TISSUE

-

24 hours of culture, into six NOD mice with spontaneous and six Lewis rats with streptozotocin (65 mg/kg)-induced diabetes mellitus. In either rodent strain overt diabetes was diagnosed for at least 3 weeks prior to transplantation (fasting blood glucose, FBG > 400 mg/dL). Mice received 5,000 (capsules = 833 to 1,250) and rats 10,000 (capsules = 1,666 to 2,500) microencapsulated islets, intraperitoneally (IP), with the animals under general anesthesia. additional

by morphometric examination of native pancreas versus isolated islets is represented by Fig 3. Retrieved tissue was comprised of over 95% pure islets, as determined by dissecting microscope, after DTZ staining (Fig 4). These islets were shown to be viable by microscopy examination after either ethidium bromide plus fluorescein diacetate or aldheyde fucsin (Fig 5) staining. Islet morphological integrity was finally established by electron microscopy examination that was associated with the presence of well-differentiated islet

Assays Insulin collected from media or acid ethanol extracts was determined by radioimmunoassay (RIA).’ Blood glucose of transplanted animals was measured by a glucose oxidase method (Beckman glucose analyzer 2, Beckman Instruments, Fullerton, CA).

cells (Fig 6).

Islet In Vitro Function Insulin release above baseline, in response to 16.5 mmol/L glucose with or without 0.1 mmol/L IBMX and to 5.5 mmol/L glucose plus 10 mmol/L arginine, was substantial and significant, as compared to baseline, and was sustained throughout 72 hours of culture maintenance, after either static incubation (Fig 7) or perifusion (Fig 8), showing that isolated porcine islets were associated with physiologic functional competence.

RESULTS

Islet Yield and Morphology Table 2 shows overall islet and insulin recoveries from 12 separate porcine pancreata, achieved by our method. We isolated an average of 11,000 islets/g of pancreas, which represents a uniquely massive yield in comparison with results previously reported. Islet-size distribution, assessed

H

isolated

k -- - i

native pancreas

n

r 1 1

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L.

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50

60

70

80

DIAMETER

90

100

110

OF ISLETS

120

130

140

(pm 1

160

160

170

180

190

200

Fig 3. Schematic reprerentation of isolated versus native pancreatic porcine-islet-size distribution after morphometric assessment.

178

CALAFIORE ET AL

Table 2. Islet snd Insulin Recoveries From Porcine P8ncre8t8 Pancreas weight (g)

80 f 8

Islet recovery (islets/g pancreas) Insulin recovery* (U/g pancreas)

11,166

f 901

1.5 f 0.2

NOTE. Mean values f SEM are shown for 12 separate pancreata. *Insulin was extracted by acid ethanol from aliquots of pure islets at the end of the isolation procedure.

Islet In Vivo Function All animals receiving intraperitoneal implants of microencapsulated porcine islets showed complete remission of hyperglycemia (Fig 9) by day 3 of treatment, which was sustained in 100% of recipients for at least 15 or 50 days in NOD mice or Lewis rats, respectively. DISCUSSION

Several methods have been reported for the isolation of islets of Langerhans from the rodent,* canine,9 porcine,’ bovine,‘Ov” and human” pancreas to validate the potential introduction of islet-cell transplantation in the therapy of insulin-dependent diabetes mellitus. So far this objective has been successfully achieved in rodentsI and in dogs,14 granting that sufficient islet-cell mass and either immunoalteration of islets or immunosuppression of recipients were provided. On the other hand, no success has been achieved yet in humans in terms of complete remission of diabetes, neither could insulin be withdrawn following islet transplan-

Fig 5. Photomicrcgrclph of freshly isol8ted Porcine islets of L8ngerh8nc after staining with aldheyde fucsin under light micrcscope (original msgnific8tion x400).

Fig 4. Photomicrogr8ph of freshly isol8ted porcine islets of Langerh8ns 8fter st8ining with diphenylthicc8rbazone. under stereomicro*cope (original m8gnification X40).

179

MASSIVE SEPARATION OF PORCINE ISLETS OF IANGERHANS

Fig 6. Electron photomicrcgraph of freshly isolated porcine islets of Lengerhanr showing well-differentiated endocrine granuli.

islet-cell

2 GLUCOSE

5.5

mM

GLUCOSE

18.5

mM

16.5

mM

+

mM

+

GLUCOSE ISMX

1

0.1

q

mM

GLUCOSE

5.5

ARGININE

10 mM

Q

p 4

0.02

@a,0 peo.01

0. L

I

I

Fig 7. Isolation release from isolated porcine islets in rasponse to insulin secretegogues after 2 hours static incubation.

I

fresh

72”h GLUCOSE 5.5

I

mM

:

16.5

mM+lBMX

0.1

mM

5.5

mM

5.5

I

II

mM + ARGMINE

10 mM

I

I O+

Fresh

120-

a 5 g

$j

?i

loo 80-

z

a *

60-

$

$j

40-

ap -

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20-

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Fig 6. Insulation secretory kinetics of isolated porcine islets during perifusion with insulin secretagogues.

I

-20

I

0

I 10

I

20

1

30

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40

TIME

,

50

1

60

,

70

(minutes)

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80

1

90

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loo

110

a

120

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130

CALAFIORE ET AL

NOD MICE [spontaneous)

LEWIS RATS (ST2 induced) t ?

I

0

10

20

JO

40

DAVS AFTER Xenotranrplantation Fig 9. lets into diabetic rodents.

50

t ?

I 60

t ?

10

1 (0

TRANSPLANT

of microencapsulated

porcine

is-

in diabetic patients.ls Although the reasons for these partial as well as transient results are still unclear, the consistency of the first clinical trials of human islet transplantation has been seemingly interdicted by the inadequacy of viable islet cell mass in combination with the occurrence of islet graft directed immune and/or adverse environmental factors eventually related to the site of implant. Recent methods have been describedI that enable the massive, semiautomated isolation of human islets; but even provided that the host’s immune reaction was circumvented, still the availability of cadaveric human donor pancreata would be insufficient for the large-scale applicability of this strategy. Hence the massive isolation and purification of suitable, tation

non-human, high-mammal islets could provide, alternatively, an attractive pancreatic tissue resource for donor islets. Porcine islets are theoretically ideal candidates, since pork and human insulins are structurally very similar and there are no restrictions in terms of availability of donor pigs. We have developed a method for the large-scale, high-yield isolation of porcine islets of Langerhans that is unique and original. In fact, previously described procedures’ reported that after the enzymatic digestion, the pig pancreas should be disrupted mechanically through choppers, macerators, or sieves to separate the islets from the pancreatic matrix. We believe that such maneuvers could be very harmful to islet viability, since porcine islets are well known to be fragile and delicate. Our method does not require any mechanical manipulation of the preparation, since pancreatic digest is already enriched in free islets that can be easily harvested from the supernatant. Furthermore, our technique provides extraordinary and reproducible high yield in pure porcine islets as it has never been achieved before and reduces significantly time-consuming operative steps. The overall time that is required for processing a porcine pancreas does not exceed 4 hours. In vitro, pretreatment immunomodulation techniques have been reported” that may result in acceptance of rodent islet grafts across histocompatibility barriers. Similar results have not been obtained yet with high-mammal islets, but intense study is in actual progress with respect to this field. Moreover, immunoisolation of islets within algin/polyaminoacidic biocompatible and semipermeable artificial membranes has been proven to successfully immunoprotect rodent islet xenografts in diabetic recipients,6.‘s thus offering a strategy that is potentially applicable also to high mammalians. We have in fact shown, preliminarily, that algin/poly-L-lysine microencapsulated porcine islets may result in full reversal of hyperglycemia in a xenogeneic system in rodents with either spontaneous or streptozotocin-induced diabetes. In conclusion, should islet immunogenicity be abrogated by either immunomodulation or immunoisolation maneuvers or both, adult porcine islets could represent an accessible and inexhaustible resource for transplantation in diabetic recipients. ACKNOWLEDGMENT

We wish to acknowledge the superb technical assistance provided to us bzy Romeo Pippi that enabled us to conduct these studies.

REFERENCES

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MASSIVE SEPARATION OF PORCINE ISLETS OF LANGERHANS

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