Patent Application
20080070301
The present invention relates to improvements in and/or relating to the treatment of diabetes using xenotransplantation. More particularly but not exclusively the present invention relates to the preparation of viable xenotransplantable porcine islets and/or the treatment of a mammalian patient (including humans) suffering from diabetes involving the transplantation into the mammal of viable porcine islets capable of producing insulin within the host and the associated use of sertoli cells within the procedure.
Type 1 (insulin-dependent) diabetes mellitus is a common endocrine disorder that results in substantial morbidity and mortality, and leads to considerable financial costs to individual patients and healthcare systems.
Treatment with insulin, while life-saving, often does not provide sufficient control of blood glucose to prevent the feared complications of the disease, which has provided the impetus for intensive research into better methods of sustaining normoglycaemia.
Among the newer treatment strategies that have been proposed, transplantation of pancreatic islet cells, obtained either from other humans or animals, has received the most attention worldwide. This is because transplantation can restore not only the insulin-secreting unit, but also the precise fine tuning of insulin release in response to multiple neural and humoral signals arising within and beyond the islets of Langerhans.
Human islet cell transplantation is limited by the shortage of human islet tissue. The use of pig islet cells is currently viewed as the most promising alternative since:
The rationale for this treatment approach (termed ‘xenotransplantation’) is that the implanted pig islets have the potential to mimic the normal physiological insulin response in type 1 diabetics such that near-normal blood glucose levels may be achievable without insulin administration or with a reduced requirement for it. As a consequence, long-term diabetes complications may be prevented and patients should experience less hypoglycaemia than they do with the currently recommended ‘intensive’ insulin regimens.
Allograft (same species transplantation) of pancreatic islets, separated from human donor organs, could induce full remission of hyperglycaemia and discontinuation of exogenous insulin treatment in patients with Type 1 diabetes mellitus. However, full success of this approach is still hampered by several problems, and in particular:
Although immune rejection is a major problem for transplantation, problems with longevity of the islet transplant unrelated to the immune system may also prevent successful islet cell transplantation. This may be ascribed to unfavourable local environmental conditions because of the heterotopic nature of the transplant site's anatomy (usually the liver) which greatly differs from the native pancreas, non specific mediators of the inflammation, -cell-induced apoptosis, or other unknown factors. Nevertheless, the extremely slow -cell turnover significantly contributes to abbreviate the functional life-span of such a differentiated tissue. Hence attempts to expand the original grafted -cell mass should be implemented.
Previous studies reporting on induction of insulin release from whole islets, which had been exposed to hormones, such as prolactin, human placental lactogen, and growth hormone, failed to demonstrate significant increase in the islet -cell replication rate1, thereby clouding the interpretation about the origin and mechanisms of the increased insulin secretion. In subsequent studies, adult human islet -cells were shown to expand during in vitro culture maintenance, within special artificial tissue matrices, and in presence of growth factors2. However, in this particular instance, the islets had been dispersed into single cells and cultured as cell monolayers. Moreover, there was no significant correlation between the increased -cell mitotic index and increase in insulin concentration. Last, the impact of islet cell aggregates, rather than whole islets, on transplantation remains unpredictable and it has not been further pursued.
In our previous work, particularly as detailed in PCT/NZ01/0009 we have disclosed the preparation and use of xenotransplantable porcine islets for the treatment of diabetes.
It is an object of the present invention to provide an improved method of preparing porcine islets which produces islets viable for xenotransplantation into a mammalian patient the islets being capable of producing insulin within a mammalian host, as well as the islet preparation so produced, or irrespectively or how produced, or a similar form.
Alternatively or additionally, it is a further object to provide an improved method of treating a mammalian patient suffering from diabetes which involves the xenotransplantation of porcine islets into the mammalian patient.
Alternatively or additionally, it is a further object to at least provide the public or medical community with a useful alternative approach to diabetes treatment.
In a first aspect the invention consists in a method of preparing a xenotransplantable porcine islet preparation capable upon xenotransplantation of producing porcine insulin in an appropriate recipient mammal, the method including or comprising:
wherein (at least some stage in the method) the islets are associated with Sertoli cells.
Preferably the islets (at least at some stage in the performance of the method) are exposed to nicotinamide.
Preferably the piglets are at −20 to +20 days of full term gestation.
Preferably the piglets are at −7 to +10 days of full term gestation.
Preferably the mammalian albumin is human serum albumin (HSA).
Preferably the collagenase is selected from human Liberase® and porcine Liberase®.
Preferably said Liberase® is human Liberase®.
Preferably the islets are treated with nicotinamide after their extraction from the pancreas.
Preferably the method includes the further step of treating the islets with IgF-1 or the N-terminal tripeptide of IgF-1 (GPE).
Preferably the pancreas and/or islets are subject to a trauma protecting agent selected from suitable anaesthetic agents.
Preferably the trauma protecting agent is Lignocaine.
Preferably the Sertoli cells are mixed with and/or co-cultured with the islets.
Preferably preparation or isolation of the Sertoli cells include use of, or exposure to a buffer to selectively reduce germinal cells.
Preferably the buffer is tris-(hydroxymethyl)-aminomethane hydrochloride.
Preferably the ratio of Sertoli cell to islets employed is substantially 10-1,000 Sertoli cells/islet.
In another aspect the invention consists in a xenotransplantable porcine islet preparation prepared according to the above method.
In another aspect the invention consists in a method of preparing an implantable device which includes at least one xenotransplantable porcine islet capable upon xenotransplantation of producing porcine insulin in an appropriate recipient mammal, said method including all comprising:
wherein nicotinamide is introduced to the islets or islet cells prior to incorporation at any one or more stages of the procedure, and
wherein, at least to the extent required to convert porcine non insulin producing islet cells to porcine insulin producing islet cells, at some stage in the procedure presenting the islets and/or islet cells to IgF-1 or the N-terminal tripeptide of IgF-1 (GPE), and
wherein (at least at some stage in the method) the islet cells are associated with Sertoli cells.
Preferably the Sertoli cells are employed within the device to substantially prevent or reduce tissue immune rejection.
Preferably the Sertoli cells are mixed with the islets, prior to insertion into, or within, the device.
Preferably preparation or isolation of the Sertoli cells include use of, or exposure to a buffer to selectively reduce germinal cells.
Preferably the buffer is tris-(hydroxymethyl)-aminomethane hydrochloride.
Preferably the Sertoli cells are co-cultured with the islets prior to incorporation.
Preferably the ratio of Sertoli cell to islets employed is substantially 10-1,000 Sertoli cells/islet.
More preferably the Sertoli cells are mixed with the islets substantially in a ratio of 10-1,000 cells/islet.
Preferably an antibiotic is used in the isolation of the islet cells.
Preferably the antibiotic is ciproxin.
Preferably the islet trauma protecting agent is Lignocaine.
In one form of the invention the device is a suitable vascularised subcutaneous collagen tube and one or more islet cells are confined within the tube.
In a second form of the invention the device is a capsule formed from a biocompatible xenotransplantable material which, in vivo, is both glucose and insulin porous, and one or more islet cells are encapsulated within the capsule.
Preferably said biocompatible material is a suitable alginate.
Preferably the alginate is in ultra pure form.
Preferably the encapsulation provides a surround which prevents, once implanted, direct tissue contact with the islets.
Preferably each encapsulation involves presenting islets and a suitable alginate solution into a source of compatible cations thereby to entrap the islets in a cation-alginate gel.
Preferably said cation alginate gel is calcium-alginate gel.
Preferably said alginate used in the solution is sodium alginate, and the islets and sodium alginate solution is presented as a droplet into a bath of suitable cations.
Preferably the islets and sodium alginate solution is of 1.6% w/w.
Preferably the islets and sodium alginate solution is presented as a droplet through a droplet generating needle.
Preferably the suitable cations are calcium chloride.
Preferably the gel encased islets are coated with a positively charged material and thereafter are provided with an outer coat of a suitable alginate.
Preferably the positive charging material is poly-L-ornithine.
Preferably the gel entrapping the islets within the outer coating is then liquefied.
Preferably the liquification involves or comes about by the addition of sodium citrate.
Preferably the encapsulation produces capsules.
Preferably the capsules contain a plurality of islet cells.
Preferably the capsules contain substantially three islet cells.
Preferably the capsules have a diameter of substantially from about 300 to 400 microns.
In another aspect the present invention comprises an implantable device being or including viable porcine islets prepared according to a method of the present invention.
In yet another aspect the invention comprises a vascularised subcutaneous tube containing viable islet cells from a −20 to +20 full term gestation piglet capable of producing porcine insulin in response to glucose within a recipient mammal,
Preferably the Sertoli cells are mixed with the islets prior to insertion into, or within, the tube. Preferably the Sertoli cells are mixed with the islets substantially in a ratio of 10-1,000 cells/islet. Preferably said culture has been of mechanically reduced harvested pancreatic tissue, such tissue having been exposed to a trauma reducing agent.
In yet another aspect the invention comprises a xenotransplantable capsule having viable islet cells from a −20 to +20 full term gestation piglet capable of producing porcine insulin in response to glucose within a recipient mammal within its biocompatible encapsulating material or materials, the capsule being such that the encapsulation is such as to prevent tissue contact with said islet cell(s) by tissue of a recipient mammal yet in vivo will allow glucose entry to the islet cells and the egress of porcine insulin from such islet cells,
Preferably the culture has been of mechanically reduced harvested pancreatic tissue, such tissue having been exposed to a trauma reducing agent.
Preferably the encapsulation has been of islet cells in the presence of a suitable antibiotic.
Preferably the Sertoli cells are mixed with the islets prior to encapsulation.
Preferably the Sertoli cells are co-cultured with the islet cells prior to encapsulation.
Preferably the Sertoli cells:islet ratio is 10-1,000 cells:islet.
In yet another aspect the invention is a method of treating a mammalian patient predisposed to or suffering from diabetes which involves the xenotransplantation into such patient at least one vascularised subcutaneous tube, capsule or implant of the present invention.
In yet another aspect the invention is a method for the treatment of a mammalian patient suffering from or predisposed to diabetes, said method including or comprising the steps of:
wherein the islets (at least at some stage in the performance of (A)) are exposed to nicotinamide;
Preferably the Sertoli cells are mixed with the islets, prior to insertion into, or within, the device.
Preferably the Sertoli cells are mixed with the islets substantially in a ratio of 10-1,000 cells/islet.
Preferably there is included also the step of administering nicotinamide to the recipient mammal prior to or after the implantation step.
Preferably the method further includes the step of prescribing for the patient, prior to or after the implantation step, a casein-free diet (as described herein).
Preferably the method further includes the step of subjecting the patient prior to or after the implantation step to a cholesterol lower drug regime.
Preferably the cholesterol lowering drug is of the “statin” family
Preferably the cholesterol lowering drug is Pravastatin® or Simvistatin®.
In one preferred form the device of step (B)(i) is a suitable vascularised subcutaneous collagen tube.
In an alternative preferred form the device of step (B)(i) is a capsule of a biocompatible, xenotransplantable material which is, in vivo, both glucose and insulin porous and one or more islet cells are encapsulated within the capsule.
Preferably the biocompatible xenotransplantable material is a suitable alginate.
According to a still further aspect of the invention there is provided a method of preparing an implant which includes at least one xenotransplantable porcine islet capable upon xenotransplantation of producing porcine insulin in an appropriate recipient mammal including the steps of:
Preferably preparation or isolation of the Sertoli cells include use of, or exposure to a buffer to selectively reduce germinal cells.
Preferably the buffer is tris-(hydroxymethyl)-aminomethane hydrochloride.
In one form the implant is (one or more) capsules within which (one or more) islets are encapsulated.
Preferably the Sertoli cells are encapsulated with the islets.
Preferably the Sertoli cells are co-cultured with the islets prior to encapsulation.
Preferably the ratio of Sertoli cell to islets employed is substantially 10-1,000 Sertoli cells/islet.
Preferably the co-culturing results in induced or increased β-cell proliferation and consequential increased endogenous insulin output.
Preferably the preparation of porcine islets includes the steps of harvesting and extracting the islets.
Preferably the porcine islets are prepared substantially according to one of the methods of preparing a xenotransplantatible porcine islet preparation hereinbefore described.
Preferably the encapsulation regime includes the steps of encapsulating the co-cultured islets and Sertoli cells within a biocompatible xenotransplantable material, the material in vivo being both glucose and insulin porous.
Preferably nicotinamide is introduced to the islets or islet cells or co-culture prior to encapsulation at any one or more stages of the procedure.
Preferably, at least to the extent required to convert porcine non insulin producing islet cells to porcine insulin producing islet cells, at some stage in the procedure the islets and/or islet cells are presented to IgF-1 or the N-terminal tripeptide of IgF-1 (GPE).
Preferably an antibiotic is associated with the islet cells.
Preferably said antibiotic is ciproxin.
Preferably said islet trauma protecting agent is lignocaine.
Preferably said biocompatible material is a suitable alginate.
Preferably the alginate is in ultra pure form.
Preferably each islet or grouping of islets is entrapped in an in vivo insulin and glucose porous biocompatible alginate or alginate like surround.
Preferably the encapsulation provides a surround which prevents, once implanted, direct tissue contact with the islets.
Preferably each encapsulation involves presenting islets and a suitable alginate solution into a source of compatible cations thereby to entrap the islets in a cation-alginate gel.
Preferably said cation alginate gel is calcium-alginate gel.
Preferably said alginate used in the solution is sodium alginate, and the islets and sodium alginate solution is presented as a droplet into a bath of suitable cations.
Preferably the islets and sodium alginate solution is of 1.6% w/w.
Preferably the islets and sodium alginate solution is presented as a droplet through a droplet generating needle.
Preferably the suitable cations are calcium chloride.
Preferably the gel encased islets are coated with a positively charged material and thereafter are provided with an outer coat of a suitable alginate.
Preferably the positive charging material is poly-L-ornithine.
Preferably the gel entrapping the islets within the outer coating is then liquefied.
Preferably the liquification involves or comes about by the addition of sodium citrate.
Preferably the encapsulation produces capsules.
Preferably the capsules contain a plurality of islet cells.
Preferably the capsules contain substantially three islet cells.
Preferably the capsules have a diameter of substantially from about 300 to 400 microns.
In a second form the implant is a vascularized subcutaneous tube within which (one or more) islets are contained.
Preferably the Sertoli cells are contained with the islets.
Preferably the Sertoli cells are co-cultured with the islets prior to encapsulation.
Preferably the ratio of Sertoli cell to islets employed is substantially 10-1,000 Sertoli cells/islet.
Preferably the co-culturing results in induced or increased β-cell proliferation and consequential increased endogenous insulin output.
Definitions
As used herein:
This invention may also be said broadly to consist in the parts, elements and features referred to or indicated in the specification of the application, individually or collectively, and any or all combinations of any two or more of said parts, elements or features, and where specific integers are mentioned herein which have known equivalents in the art to which this invention relates, such known equivalents are deemed to be incorporated herein as if individually set forth.
The invention consists in the foregoing and also envisages constructions of which the following gives examples.
Preferred forms of the present invention will now be described with reference to the accompanying drawings in which:
1.General
The present invention recognizes the ability to source appropriate islets from piglets which have similar structural similarities of insulin to humans, and similar physiological glucose levels to humans. The piglets used are at or near full term gestation. The islets are converted into an appropriate xenotransplantable source of islets with viability in a human being by following certain procedures in respect of the harvesting and extraction of the islets, the treatment of the islets prior to xenotransplantation as well as regimes of use of such islets.
The major advantage of porcine islet cell transplantation over human islet cell transplantation is that the islet cell source can be readily expanded, and the biosafety of the cells can be thoroughly explored prior to transplantation. From a practical viewpoint, pancreas removal and islet cell isolation can be performed expeditiously in an ideal environment.
Important considerations relevant to the use of porcine islet cells in transplantation approaches for type 1 diabetes include the following:
2. The Nature of the Disease Causing Diabetes
Successful long-term allotransplantation of human islets can be achieved in over 80% of patients when the disease is caused by non-immune processes. In contrast, even islets obtained from a non-diabetic twin cannot reverse autoimmune diabetes long-term in the diabetic twin member. This emphasises the critical role of autoimmunity in the failure of islet transplantation. This observation has been validated in allotransplantation of rodents with diabetes caused by autoimmunity as compared with diabetes due to pancreatectomy or chemical cell destruction. No large animal model of autoimmune diabetes exists. It is possible that the use of islets from different species (xenotransplantation) could avoid autoimmune destruction of transplanted islets, as the immune process of xenotransplant rejection is different to that of allotransplant rejection, but this is entirely hypothetical in humans.
3. Isolation and Preparation of Porcine Islet Cells for Xenotransplantation
3a. Animal Source and Transportation
All animals intended as a source of pancreatic tissue for xenotransplantation are obtained from a specific pathogen-free (SPF) pig breeding facility which is maintained in accordance with the American Association for Accreditation of Laboratory Animal Care (AAALAC). The facility maintains a high-health status colony with excellent standards of husbandry, and operates a record system that is readily accessible and archived indefinitely. Donor sows and sires are selected with the underlying objective of producing strong heterosis in donor litters.
3b. Isolation and Purification of Islet Cells
Following surgical removal, the donor pancreases are transferred to a cleanroom facility for further processing in a cold plastic container in 50 ml tubes containing cold Hanks' Balanced Salt Solution (HBSS) with 0.2% human serum
3c. Digestion
The islet cells are isolated by standard collagenase digestion of the minced pancreas via the procedure documented by Ricordi et al. (1990), though with some modifications. Using aseptic technique, the glands are distended with Liberase™ (1.5 mg/ml), trimmed of excess fat, blood vessels and connective tissue, minced, and digested at 37° C. in a shaking water bath for 15 minutes at 120 rpm. The digestion is achieved using lignocaine mixed with the Liberase™ solution to avoid cell damage during digestion. Following the digestion process, the cells are passed through a sterile 400 mm mesh into a sterile beaker. A second digestion process is used for any undigested tissue.
We have determined that much greater yields per neonatal pig pancreas can be obtained using either pig or human Liberase™ (eg; sourced in New Zealand from Roche) rather than collagenase. Whilst there is disclosure in “Improved Pig Islet Yield and Post-Culture Recovery Using Liberase P1 Purified Enzyme Blend”, T J Cavanagh et al. Transplantation Proceedings 30, 367 (1998) and in “Significant Progress In Porcine Islets Mass Isolation Utilizing Liberase HI For Enzymatic Low-Temperature Pancreas Digestion”, H. Brandhorst et al. Transplantation Vol 68, 355-361 No. 3, Aug. 15, 1999 the yields therefore therein are low compared to those we have discovered. If, for example, in following the procedure of Brandhorst et al. there is a yield increase of islets over collagenase of from 400 to say 800 with the procedure using human Liberase™ (ie; Liberase™ HI) as in the Brandhorst et al. procedure but confined to neonatal porcine islets such as those as 7 days post delivery extra ordinarily larger yields are possible, namely, the equivalent to from 400 which would be the case with crude collagenase to 30000 which as can be seen as very much greater than that to be expected from following the procedure of Brandhorst et al. with pigs.
3d. Washing and Culture
The digested tissue is washed three times, and seeded into cell culture media RPMI 1640 to which is added 2% human serum albumin (HSA), 10 mmol/L nicotinamide, and antibiotic (Ciproxin).
3e. Quality Control Procedures
To exclude any contamination of the tissue, quality control procedures are undertaken on cell culture samples after isolation and before encapsulation (further details are given in SOP P101). Three days after isolation, the cell culture is tested for microbiological contamination by accredited laboratories. Testing for porcine endogenous retrovirus (PERV) is undertaken in our laboratory. The islet yield is determined via dithizone (DTZ) staining of the cells, as specified in SOP Q200. Dithizone is a zinc-chelating agent and a supravital stain that selectively stains zinc in the islets of Langherhans, producing a distinctive red appearance.
The viability of the islet cells is determined using acridin orange and propidium iodide, as specified in SOP Q201. Acridin orange is a fluorescent stain that readily passes through all cell membranes to stain the cytoplasm and nucleus. Bright green fluorescence in both the nucleus and cytoplasm on exposure to ultraviolet (UV) light denotes intact live cells. Conversely, propidium iodide is a fluorescent stain that cannot pass through an intact membrane. It emits a bright red fluorescence when exposed to UV light, and the presence of propidium iodide in a cell nucleus indicates severe damage or a dead cell.
3f. Determination of in vitro Insulin Secretory Capacity
Static glucose stimulation (SGS) is used to assess in vitro function of the porcine islets by exposing them to low and high concentrations of glucose and theophylline. Determination of the in vitro insulin secretory capacity is undertaken on both free islets (after 3 days in culture) and after their subsequent encapsulation or confinement.
4. Xenotransplantation
4a. The Viability of the Islets for Xenotransplantation
The processes by which islets are purified prior to transplantation are traumatic to these highly specialised tissues. Such trauma can induce necrosis or apoptosis—the latter being quite delayed.
Further trauma may result from encapsulation. Processes used by us in both the preparation of islets and their encapsulation have been optimised to ensure minimal damage to the islets. Such procedures have ensured zero warm ischaemia (compared with hours with most human islet preparations), have involved the use of nicotinamide to enhance successful in vitro explanation, have involved minimal incubation time with collagenase or liberase, have involved swift non-traumatic encapsulation technology, have involved the use of IgF-1 (or the GPE tripeptide thereof), the use of an anaesthetic such as lignocaine, and the use of an antibiotic such as ciproxin etc.
Our preferred preparation preferably uses neonatal (7-day old) islets which is crucial in both limiting islet trauma during purification, and assuring sufficient maturation of the islets for stimulated insulin production.
The IgF-1 (Human Insulin-like Growth Factor I) is used in order to induce immature porcine islets to mature to their insulin-producing form. IgF-1 is a potent mitogenic growth factor that mediates the growth promoting activities of growth hormone postnatally. Both IgF-1 and IgF-2 are expressed in many cell types and may have endocrine, autocrine and paracrine functions. The preferred form of IgF-1 we have found to be the amino-teminal tripeptide glycine-proline-glutamate of IgF-1 (GPE).
4b Vascularised Subcutaneous Collagen Tube Procedure.
The islets may be implanted in a suitably vascularised subcutaneous collagen tube using Sertoli cells as the method of preventing tissue immune rejection.
In brief a closed ended tube of stainless steel mesh containing a loosely fitting Teflon rod is inserted subcutaneously in the intended graft recipient. Six weeks later the rod is removed—leaving a highly vascularised tube of collagen. A mixture of islets prepared as above together with Sertoli cells prepared by the method of Rajotte from the testes of the same piglets from whom the islets were obtained. The Sertoli cells are mixed with the islets in a ratio of about 10 to 1,000 cells/islet and inserted into the vascular tube. Which is then sealed with a Teflon stopper.
The effect of such a device containing piglet islets (approx 300,000)+Sertoli cells on insulin requirement in a 15 year old human insulin dependent diabetic subject is shown in
The secretion of porcine C-peptide in response to IV glucose in this subject 4 weeks after transplantation is shown in the
4c. Alginate Encapsulation Procedure
Sodium alginate used for this procedure is extracted from raw material sources (seaweed) and prepared in a powdered ultrapure form. The sterile sodium alginate solution (1.6%) is then utilised at the Diatranz Islet Transplant Centre to manufacture encapsulated islets. The encapsulation procedure (University of Perugia) involves extruding a mixture of islets and sodium alginate solution (1.6%) through a droplet generating needle into a bath of gelling cations (calcium chloride). The islets entrapped in the calcium-alginate gel are then coated with positively charged poly-L-ornithine followed by an outer coat of alginate (0.05%). The central core of alginate is then liquefied by the addition of sodium citrate. Most capsules contain 3 islet cells and have a diameter of 300 to 400 μm.
The encapsulated islets are kept in cell culture, and then checked for contamination, insulin release and viability before transplantation. They are only released for transplantation if all quality control tests are negative.
4d. Drugs Used in the Recipient
Transplantation does not require and avoids the need for cytotoxic agents to suppress the immune system. Such agents are able to enter the alginate microcapsule and cause islet toxicity, as well as causing systemic toxicity. Instead, nicotinamide and a special diet are used.
The transplantation procedures of our earlier patent specification have the ability over a period prior to rejection of providing porcine insulin. In this respect, we ourselves conducted clinical trials.
Four type 1 diabetic adolescents received 10,000 free islets/kg bodyweight by intraperitoneal injection. The islets were located from term piglets using the standard collagenase digestion, purification and culture techniques described in section 3.2. All four recipients received oral nicotinamide (1.5 g/day) and a casein-free as herein defined diet both pre- and post-transplantation. A prompt reduction in insulin requirements, which was not clearly dose-related, was noted in the first week after transplantation. The reduction in insulin dosage range from 21 to 32%, and the response lasted for up to 14 weeks. However, insulin doses subsequently returned to their previous levels.
The most likely reason for the transplant failure in these patients was chronic rejection. However, no adverse effects were noted.
We have shown alginate encapsulation is a suitable method for limiting such chronic rejection. We have shown alginate-encapsulated porcine islet cell transplants in two human diabetic patients, prolonged functioning of the transplants. The islets were transplanted by intraperitoneal injection, one patient receiving 15,000 IEQ/kg (total 1,300,000 islets) and the other 10,000 IEQ/kg (total 930,000 islets). Both patients were treated pre- and post-transplantation with oral nicotinamide and a soy-based/casein-free as herein defined diet.
The preferred procedure as shown in
4e. Use of Sertoli Cells
The present invention is directed to dealing with the islet transplant mass problem. Islet -cells originally derive from both differentiation of pancreatic exocrine duct stem cells, and replication of pre-existing, already differentiated cells. The -cell proliferation activity varies upon age (from 10% of the fetal period to 3% of adulthood), although specific factors or conditions may influence and/or promote growth of these cells at any time, and in particular:
However, it is quite unlikely that, within a transplant setting, under putative unfavourable environmental conditions, the -cells undergo significant replication. The latter has been observed in alginate-microencapsulated islets, although the death rate clearly surpassed the islet cell mitotic rate, thereby leading to progressive loss of the original transplanted cell mass.
To deal with this problem, ancillary “nursing” cells, associated with cell growth induction capacity might result in prolongation of the islet -cells survival and functional competence.
In the present invention, Sertoli cells have been employed as a potential “nursing” cell system for the islets. Sertoli cell functional properties, and in particular synthesis of growth factors such as IGF-I, transforming growth factor , endothelial growth factor, and clusterin, favour the -cell expansion, while on the immunity front, expression of Fas-L provides the islet transplant with immunoprotection.
In accordance with the invention Sertoli cells upon in vitro co-culture with homologous, isolated whole rat pancreatic islets, could alter the -cell low proliferative cycle, by reversing the adult elements into foetal-like status has been examined. This would coincide with significantly enhanced -cells mitogenicity.
In the specific instance of Sertoli Cells, production of several different growth factors is coupled to expression of molecules, such as Fas-L, that are associated with immune properties.
Theoretically, Sertoli Cell-derived Fas-L could induce apoptotic destruction of the tissue transplant-directed CD4+ and CD8+ Tc populations. Furthermore, Sertoli Cells in their original anatomic situation, are part of the so called “hemato-testicular barrier”. Therefore the Sertoli Cell-related immunoprivileges might be possibly transferred to the islet cell system. Sertoli Cell-related cell growth induction capacity, in conjunction with immunomodulatory activity, have been proven to succeed in preliminary experimental studies. Cerebral grafts of Sertoli Cell relieved symptoms in rats with pharmacologically-induced hemiparkinsonism. Moreover, allograft of unpurified Sertoli Cells in combination with islets, beneath the kidney capsule of rats with streptozotocin-induced diabetes, significantly prolonged the achieved normoglycemia, as compared to controls receiving islet Transplant alone.
Our studies have shown that Sertoli Cells, at proper concentrations, are able to promote significant -cell replication when co-incubated with homologous islets. Additionally, the increase in -cell mitotic activity results in an associated significant increment in endogenous insulin output, both in standard culture conditions, and under glucose stimulation, as compared with Sertoli Cell-free control islet cells.
Our studies have demonstrated that:
Porcine islets in culture which were exposed to IgF-1, increased their insulin response to glucose, by up to a 3-fold increase.
A concentration of 0.1 ug/ml IgF-1 in culture is sufficient to produce optimal insulin secretion during glucose challenge. No further benefit was achieved by increasing the concentration of IgF-1.
Variations on the duration of IgF-1 exposure were tried on the porcine islet cells. However no increased benefit was found on culturing the islets with IgF-1 beyond a 24 hrs period, post isolation.
This increased insulin production persisted to 14 days post IgF-1 exposure. Longer durations are yet to be investigated.
Withdrawal of Nicotinamide from the culture media eliminated the benefit of IgF-1 on islet insulin production.
A concentration of 0.1 ug/ml IgF-2 during culturing appeared to increase insulin production of porcine islet cells, after an initial exposure of 24 hrs. However, this increase was transient to 3 days post exposure.
Prolonged exposure to IgF-2 beyond 24 hrs, failed to increase the insulin production of the islet cells in response to glucose.
GPE is a tripeptide (gly-pro-glu) derived from IGF-1. It is a novel neuroactive peptide with a potent effect on acetylcholine and dopamine release in cortical slices. The studies done using GPE support the concept that the proteolytic products of the IGF-1 precursor play a role in the regulation of brain functions.
The aim of this example was to present the effect of GPE on the function of isolated porcine islets in vitro.
Method
Keep the cells 3 days in culture before Static Glucose Stimulation (SGS). SGS involves exposure of the cells to low and high concentration of glucose to check insulin production. Using 0.1 ug/ml concentration add IGF1 and GPE to two plates 24 hours before SGS (day 2 after isolation)
Results
Exposure of neonatal porcine islets in culture to GPE increased the insulin response to glucose up to 11.5 fold compared with the control cells.(Stimulation Index control 13.3 compared to 24.8 when GPE was used) Viability of the cells was >85% DTZ, AO/PI staining)
A concentration of 0.01 ug/ml of GPE in culture is sufficient to produce optimal response during glucose challenge. No further benefit was achieved by increasing the concentration of GPE in culture. See figure below.
The results suggest that GPE could be used during porcine islet cell culture to improve the quality and function of the cells before transplantation. Furthermore GPE is a novel neuroactive peptide found in human brain.
Lidocaine is a membrane stabiliser and phospholipase A2 inhibitor. When used at a 1 mM concentration during Collagenase digestion of 7 d old porcine pancreas, a 2-fold increase in islet yield is produced.
Islet endocrine function was assessed after 3 days in culture via static glucose stimulation. Islets isolated with Lidocaine during digestion produced a 3-fold increase in insulin secretion in response to glucose challenge.
Conclusion: The use of Lidocaine during pancreatic digestion increases the insulin production/g of pancreas by 6-fold.
Freshly prepared neonatal pig islets were prepared by standard isolation procedure and cultured for two days in RPMI medium with standard additions.
Streptomycin (100 mg/ml)and Penicillin (100 U/ml) were included in one flask and Ciproxin (3 mcg/ml) in another.
The islets were harvested and an aliquot subjected to stimulation with theophylline and high glucose.
The comparative insulin release from the islets—a measure of viability is shown in the
Pancreases of neonatal piglets aged 7 days were obtained as above and islets extracted by the same process, varying only the source and amount of collagenase. The yield/gram of pancreas is shown in the Figure.
Islets extracted using these variations in collagenase source and amount were assessed for viability using propidium iodide and dithizone for insulin content.
The islets were then assessed for functionality by static glucose stimulation as above. The results are shown in the Figure below.
It is apparent that the Liberase™ preparations at suitable concentrations gave higher yields and function in vitro than the previously optimised concentration of Collagenase P.
Islets prepared with the best concentration of Liberase P and H in this way were injected intraperitoneally into CD1 mice made diabetic by intravenous streptozotocin. The dose used was 10 islets/g body weight of mouse. Ten days after such treatment the number of mice no longer diabetic was assessed.
1/7 of the mice treated with the islets isolated with Liberase P and 4/7 of those islated with Liberase H were non diabetic.
Similar experiments were performed using spontaneously diabetic NOD mice. Of the surviving mice at 10 days after transplantation 3/7 of the Liberase P treated islets and 3/3 of the Liberase H islets were no longer diabetic
The encapsulation procedure has been developed by Dr R Calafiore at the University of Perugia. The novel medium size microcapsules (300-400μ MSM) are prepared by atomizing the islet-alginate suspension through a special microdroplet generator.
Sodium alginate used for this procedure is extracted form raw material sources (seaweed) and prepared in powdered ultrapure form (Keltone LVCR).
The encapsulation procedure involves extruding a mixture of islets and sodium alginate solution (1.6%) through a droplet generating needle into a bath of gelling cations (calcium chloride). The islets entrapped in the calcium-alginate gel are then coated with positively charged poly-L-ornithine followed by an outer coast of alginate (0.05%). The central core of alginate is then liquefied by the addition of sodium citrate. Most capsules contain 3 islet cells and have a diameter of 300 to 400 μm.
The encapsulated islets are kept in cell culture, and then checked for contamination, insulin release and viability before transplantation.
The following examples outline specific materials, methods and procedures which have been employed. As would be known by those skilled in the art, other suitable techniques or materials, or variations on the following techniques or materials may be employed without departing from the scope of the invention.
A) Basic Methodologies
1) Pancreatic Islet Source and Isolation Procedure
Islets were isolated and purified from adult Sprague Dawley male rats, weighing approximately 150 g., according to the following method:
Upon laparotomy, the common bile duct was cannulated by a polyethylene catheter, and ligated prior to its merging into the small bowel. A collagenase solution was manually delivered into the duct so as to induce retrograde distention of the pancreas. The distended pancreas was gently detached from the small and large bowel, and finally the stomach, so as to avoid any discontinuation of the organ capsule, which would cause collagenase's leakage and pancreas deflation, with compromission of the digestion process. Upon removal of the spleen and lymphonodes, the pancreas was placed in 37 C bath, and shaken (130-140 cycles/min.) until the tissue came finely apart. The tissue digest, upon wash in Hank's balanced salt solution (HBSS) was centrifuged against discontinuous Eurocollins+Ficoll density gradients, at 400 g for 25 min. at 4 C. A pure islet cell band was detected and recovered at the 1.096/1.060 density interface. The average absolute yield in islet equivalents (IEQ, islets quoted as they all measured 150 μm in diameter) per pancreas was 600.
2) Sertoli Cell Source and Retrieval Procedure
Sertoli Cells were separated from the testes of prepubertal (15 days old) SD rats, according to the following adaptation of prior art methods.
Each testicle pair was stored, soon after retrieval, in cold Eurocollins for 30 minutes. Under sterile conditions, the testes were finely minced until a fine homogeneous tissue was obtained. The tissue underwent multiple-step enzymatic digestion by collagenase P, trypsin, and DNAase. The digest, after several washes in TCM 199, was resuspended in HAM F12 supplemented with nicotinamide, 3-isobutyl-1-methylxanthine, serum albumin and pen-strep. The tissue suspension was finally filtered through 500 μm pore-size stainless mesh, plated in 100×15 mm. Petri dishes and culture-maintained at 37 C in 5% air/CO2 for 48 hours (HAM F-10, supplemented with Nicotinamide, IBMX, 10% Fetal Bovine Serum, 110 mg/dl glucose).
Finally, the tissue was treated with 20 mM tris-(hydroxymethyl)-aminomethane hydrochloride buffer to eliminate any residual germinal cells. Final yield of the procedure was 60 millions of 90% pure Sertoli Cell per testicle. Sertoli Cell morphological characterization and identification was conducted by staining the preparation with Sudan III, and anti-vimentin MoAb, while cell viability was documented by Trypan Blue exclusion staining (the cells were incubated for 15 minutes at room temperature, in the presence of 0.2% Trypan Blue in HBSS). Additionally, cell viability was also assessed by staining with ethidium bromide and fluorescein diacetate, under fluorescence microscopy. Only the Sertoli Cell preparations that scored over 90% viability entered the study protocols. At the end of the separation process, to detect eventual contaminating Leydig cells, the tissue suspension was cyto-enzymatically stained to assess the activity of 3-hydroxy-steroidodehydrogenase (staining with Nitro-Blue Tetrazolium), an enzyme that is specifically associated with this cell type. Presence of Leydig cells was negligible and they completely disappeared by 48 hrs. of culture maintenance. Last, Sertoli Cell functional competence was shown by the in vitro capability of these cells to convert testosterone enanthate into 17-estradiol in presence of follicle-stimulating hormone: the assay's specificity was granted by the fact that FSH receptors are selectively expressed by Sertoli Cells. All hormones were measured by R.I.A. (intra-assay c.v.=3.2-4.5%; inter-assay c.v.=4-8%)
B) In Vitro Studies
1) Islet-Sertoli Cell Co-Incubation
Batches of 20 islets were incubated with either 200000 (islet+Sertoli Cell 200000) or 400000 Sertoli Cell (islet+Sertoli Cell 400000), in HAM F-12, in quadruplicate. Isolated islets or Sertoli Cell batches served as controls. Tissue culture medium was changed every 3 days and the supernatant was stored at −20 C. At 11 days of co-culture, the study was terminated, and the samples were processed for examination under confocal laser scanning microscopy (CLSM). Insulin (IRI) levels were determined by RIA (itra-assay c.v.<5.5%; inter-assay c.v.=9.0%). To assess the islet functional performance, the batches were sequentially exposed to glucose at different concentrations (50-300-50 mg/dl) in a 2 hr. static incubation system.
2) Microscopic Studies
a) Sample Processing
The cell preparations were treated following adaptations of prior art procedures:
Bromo-deoxyuridine (BrdU) was added to the culture medium to a final concentration of 10 μM, for 24 hours of culture. Subsequently, the islets were washed with PBS and fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.0) for 30 min. at room temperature. The BrdU-labelled islets, upon denaturation of DNA by acid hydrolysis (2M HCl), and following washes, were incubated with a 1:25 dilution of a mouse MoAb anti-BrdU (anti-BrdU mouse MoAb) in PBS with 0.3% Triton X-100 (PBS/T) for 18 hours on a rotating plate, at room temperature. After wash, the islets were then incubated with a 1:25 dilution of fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse immunoglobulin G in PBS/T for 18 hours on a rotating plate. After wash, the islets were stored in PBS/T and thereafter prepared (aqueous mounting, immuno-Fluore mounting medium) for CLSM examination. For double labelling experiments the islets were finally incubated with mouse anti-insulin Ab for 18 hours at 4 C, and with anti-mouse-TRITC, for 18 hours at 4 C, as a secondary Ab.
b) Confocal Laser Microscopy (CLSM) Examination
Samples were imaged by a LSM410 microscope. This confocal system was coupled with a 25 mW multiline Argon ion laser and a 1 mW HeNe ion laser as light sources, used to reveal FITC signals with 488 nm, and TRITC signal with a 543 nm wavelength, respectively. Samples were examined with a ×100, 1.3 numerical aperture PlanNeofluar objective lens. Images were acquired, frame by frame, with a scanning mode format of 512×512 pixels. The HeNe laser, which produces a major line at 543 nm, was employed for double labelling experiments. To this end, FITC and TRITC signals were separated by a secondary dichroic mirror (565 nm) and simultaneously detected by two photomultiplier tubes. A band pass filter (520±15 nm for FITC) and a barrier filter (590 nm for TRITC) were placed before the two photomultiplier tubes to avoid overlapping between the two signals, as described in the prior art.
c) Image Processing Analysis
Digitalized optical sections, ie Z series of confocal data (“stacks”) were transferred from the CLSM to the graphics workstation Indigo Irix XS24 and stored on the graphics workstation with a scanning mode format of 512×512 pixels and 2567 grey levels. The image processing was performed using the ImageSpace software (Molecular Dynamics), as previously described.
To reduce the unwanted background noise generated by the photomultiplier signal amplification, all the image stacks were treated with a three-dimensional filter (Gaussian filtering) that was carried out on each voxel, with a mask of 3 pixel in the x, y and z direction (3×3×3). The FITC and TRITC or PI signals were elaborated to optimize the contrast, the brightness and the intensity of the images.
To analyse the colocalization of BrdU and insulin, an ImageSpace software tool that creates a two-dimensional scatter plot diagram was employed. It shows in a third colour how dual labels are spatially distributed or co-localized. In the co-localization scatter plot it has been detected a cluster corresponding to the areas of the specimen recorded by both detectors. Within this cluster we have selected the area with high pixel values, to avoid background interference. The resulting image could be superimposed on both fluorescence or transmitted (phase contrast) images. For a better visualization we have employed a phase contrast image to show cell topography and the sites of colocalization in green. Photographs were taken by a digital video recorder Focus ImageCorder Plus using 100 ASA Tmax black and white film or 100 ASA Kodacolor Gold film.
To assess positivity of the BrdU-labelled nuclei, at least 40 fluorescent brilliant spots corresponding to DNA replicon clusters were counted, independently of the exhibited fluorescent morphology. To assess the percentage of replicating cells, all the insulin-labelled cells were counted, by assembling the optical sections from confocal microscopy analysis, through the entire islet. Finally, the BrdU-labelled cells were related to the islet cells they belonged to. Confocal laser microscopy analysis excluded that all cells were counted more than once.
C) In Vivo Studies
The same Sertoli Cell and islet tissue batches employed for the in vitro studies were used to graft CD-1 mice, weighing approximately 25 g, that had been rendered diabetic by low dose, multiple injections of streptozotocin (STZ), at 40 mg/kg/d, for five days.
Blood glucose levels were measured at 10 days of the last STZ injection, in post-absorptive conditions. Only mice that were associated with blood glucose values in excess of 400 mg/dl during at least 3 consecutive measurements entered the study. “Transplant survival” was associated with reversal of hyperglycaemia and maintenance of blood glucose lower than 150 mg/dl. Animals that returned to hyperglycaemia after the initial remission were assumed to fail. 10 diabetic mice were divided into two groups:
Blood glucose was measured by a reflectometer at 3 and 7 days of transplant, and thereafter once a week throughout the study.
The microcapsules were prepared according to our method where islet containing AG gel beads were coated with PLO and an outer layer of AG, so as to provide the islets with an immunoisolatory shield.
D. Statistical Analysis
Confocal Microscopy Studies
Data were the mean of three different experiments and were expressed as mean±SD. The asterisk indicates significant differences (p<0.01) in a Student's paired t test. All of the other differences were found to be not significant with p>0.01.
In vitro Metabolic Studies
E. Results
1) Assessment of -Cell Mitotic Activity Upon Co-Culture with Sertoli Cells
With reference to
The presence of replicating cells within the islets with high insulin expression was examined using double labelling experiments with CLSM to obtain on the same focal plane, within the same cell, the fluorescence derived from insulin and from BrdU
As illustrated in
2) Quantification of -Cell Mitotic Figures
To assess the percentage of pancreatic -cells entering the S-phase of the cell cycle upon Sertoli Cell co-culture, we used labelling with 5′-BrdU, followed by fluorescent immunostaining. The results of these experiments are shown in Table 1.
*p values < 0.01 were significant.
Cell proliferation was assessed at 24 hours after mitogenic stimulation, since it had previously been reported that this is the time, after a mitogenic stimulus, when the highest number of cells, that have entered S-phase, is detectable. While under control conditions no more than 1% of 5′-BrdU positive -cells were evidenced, the percentage of the -cell-related mitotic figures significantly raised from 1% to 8.1% upon islet cell-Sertoli Cell co-incubation.
3) In Vitro Metabolic Data
Consistent with the above reported morphological findings, we have observed significant increase in endogenous insulin output from the islets that were co-cultured with Sertoli Cells, in comparison with either islets or Sertoli Cells alone, throughout the time period of in vitro culture maintenance (total IRI levels from 90±10 μU/ml—islets alone—to 127.5±12 μU/ml—islets+Sertoli Cells, p<0.05). The enhanced insulin secretion, deriving from the islet-Sertoli Cell complexes, as compared to I alone, under static glucose stimulation, on both days 3 (
4) In Vivo Transplant Data
Reversal of hyperglycaemia was fully achieved in both experimental groups and sustained in 100% of the recipients, at 45 days post-transplant, while at 52 days of graft one animal of the group (Islet), against none of the (Islet+Sertoli Cell) group failed. The first animal belonging to the (I+Sertoli Cell) group showed recurrence of hyperglycaemia at day 75 post-transplant. At that time, 3/5 animals of the group (Islet) had failed. At day 100 of graft, three animals of group (Sertoli Cell+Islet) against one from group (Islet) were still euglycemic.
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| Number | Date | Country | Kind |
|---|---|---|---|
| 507616 | Oct 2000 | NZ | national |
| 507963 | Nov 2000 | NZ | national |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 10398744 | Jun 2003 | US |
| Child | 11775797 | Jul 2007 | US |
| Parent | PCT/NZ01/00228 | Oct 2001 | US |
| Child | 10398744 | Jun 2003 | US |
