CHAMBER FOR ENCAPSULATING SECRETING CELLS

Abstract

The invention relates to an encapsulating chamber for secreting cells, comprising a closed shell made of a semi-permeable membrane, said membrane comprising at least one layer of porous biocompatible polymer, and one layer of non-woven biocompatible polymer.

Claims

1.-17. (canceled)

18. A method for treating a patient in need thereof, comprising the step of implanting a bioartificial organ in the body of the patient, wherein the bioartificial organ comprises a chamber comprising a closed shell made of a semi-permeable membrane, delimiting a space capable of containing said secreting cells producing at least one substance of therapeutic interest, wherein said membrane comprises a layer of biocompatible non-woven polymer located between two layers of porous biocompatible polymers, wherein the chamber encapsulates secreting cells producing at least one substance of therapeutic interest for the patient.

19. The method of claim 18, wherein said membrane consists of a layer of biocompatible non-woven polymer located between two layers of porous biocompatible polymers.

20. The method of claim 18, wherein said non-woven biocompatible polymer is chosen from the group consisting of polycarbonate (PC), polyethyleneimine, polypropylene (PP), poly(ethylene terephthalate) (PET), poly(vinyl chloride) (PVC), polyamide, polyester and polyethylene (PE).

21. The method of claim 18, wherein said porous biocompatible polymer of at least one layer is chosen from the group consisting of polycarbonate (PC), polyethyleneimine, polypropylene (PP), poly(ethylene terephthalate) (PET), poly(vinyl chloride) (PVC), polyamide, polyester and polyethylene (PE).

22. The method of claim 18, wherein at least one, or the two layer(s) of porous biocompatible polymer is (are) made hydrophilic by surface physical or chemical modification, and covered with at least one hydrophilic polymer.

23. The method of claim 18, wherein one of the two layers of porous biocompatible polymers has a pore density of between 10.sup.6 pores/cm.sup.2 and 10.sup.11 pores/cm.sup.2.

24. The method of claim 18, wherein the total thickness of the membrane is between 45 m and 200 m.

25. The method of claim 18, wherein the thickness of one of the layers of biocompatible polymer is between 5 and 40 m, and the thickness of the other layer of biocompatible polymer is between 25 and 100 m.

26. The method of claim 18, wherein the internal diameter of the pores present on one of the layers of biocompatible polymer is between 5 and 100 nm, and the internal diameter of the pores present on the other layer of biocompatible polymer is between 100 and 2000 nm.

27. The method of claim 22, wherein at least one layer is covered with a hydrophilic polymer which contains at least one biologically active molecule.

28. The method of claim 18, further comprising a biocompatible sheet contained in said shell, said sheet optionally comprising projections at its surface.

29. The method of claim 19, wherein the layer external to the shell has pores with an internal diameter of between 100 and 2000 nm, and the layer internal to the shell has pores with an internal diameter of between 5 and 100 nm.

30. The method of claim 18, wherein the chamber comprises at least one connector which makes it possible to establish a communication between the exterior and the interior of the shell.

31. The method of claim 18, wherein the chamber is circular and has a diameter of between 3 cm and 20 cm.

32. The method of claim 18, wherein said non-woven polymer is polyester.

33. The method of claim 18, wherein said porous biocompatible polymer of at least one layer is polyester.

34. The method of claim 18, wherein the internal diameter of the pores present on one of the layers of biocompatible polymer is between 5 and 100 nm, and the internal diameter of the pores present on the other layer of biocompatible polymer is between 200 and 1000 nm.

35. The method of claim 18, wherein the patient has diabetes and the encapsulated cells secrete insulin.

36. The method of claim 18, wherein the bioartificial organ is implanted in the intraperitoneal cavity or in the extraperitoneal space.

37. The method of claim 18, wherein the bioartificial organ comprises at least one connector and a tube connected to the connector, the method further comprising filling and emptying the bioartificial organ to renew the cells.

38. The method of claim 19, wherein said non-woven biocompatible polymer is chosen from the group consisting of polycarbonate (PC), polyethyleneimine, polypropylene (PP), poly(ethylene terephthalate) (PET), poly(vinyl chloride) (PVC), polyamide, polyester and polyethylene (PE).

39. The method of claim 19, wherein said porous biocompatible polymer of at least one layer is chosen from the group consisting of polycarbonate (PC), polyethyleneimine, polypropylene (PP), poly(ethylene terephthalate) (PET), poly(vinyl chloride) (PVC), polyamide, polyester and polyethylene (PE).

40. The method of claim 19, wherein at least one, or the two, layer(s) of porous biocompatible polymer is (are) made hydrophilic by surface physical or chemical modification, and covered with at least one hydrophilic polymer.

41. The method of claim 19, wherein one of the two layers of porous biocompatible polymers, has a pore density of between 10.sup.6 pores/cm.sup.2 and 10.sup.11 pores/cm.sup.2.

42. The method of claim 19, wherein the total thickness of the membrane is between 45 m and 200 m.

43. The method of claim 19, wherein the thickness of one of the layers of biocompatible polymer is between 5 and 40 m, and the thickness of the other layer of biocompatible polymer is between 25 and 100 m.

44. The method of claim 19, wherein the internal diameter of the pores present on one of the layers of biocompatible polymer is between 5 and 100 nm, and the internal diameter of the pores present on the other layer of biocompatible polymer is between 100 and 2000 nm.

45. The method of claim 40, wherein at least one layer is covered with a hydrophilic polymer which contains at least one biologically active molecule.

46. The method of claim 19, wherein the chamber further comprises a biocompatible sheet contained in said shell, said sheet optionally comprising projections at its surface.

47. The method of claim 19, wherein the layer external to the shell has pores with an internal diameter of between 100 and 2000 nm, and the layer internal to the shell has pores with an internal diameter of between 5 and 100 nm.

48. The method of claim 19, wherein the chamber comprises at least one connector which makes it possible to establish a communication between the exterior and the interior of the shell.

49. The method of claim 19, wherein the chamber is circular and has a diameter of between 3 cm and 20 cm.

50. The method of claim 19, wherein said non-woven polymer is polyester.

51. The method of claim 19, wherein at least one of the layers of porous biocompatible polymer of is polyester.

52. The method of claim 19, in which the internal diameter of the pores present on one of the layers of biocompatible polymer is between 5 and 100 nm, and the internal diameter of the pores present on the other layer of biocompatible polymer is between 200 and 1000 nm.

53. The method of claim 19, wherein the patient has diabetes and the encapsulated cells secrete insulin.

54. The method of claim 19, wherein the bioartificial organ is implanted in the intraperitoneal cavity or in the extraperitoneal space.

55. The method of claim 19, wherein the bioartificial organ comprises at least one connector and a tube connected to the connector, the method further comprising filling and emptying the bioartificial organ to renew the cells.

Description

DESCRIPTION OF THE FIGURES

[0142] FIG. 1: Permeability of poly(ethylene terephthalate) (PET) or polycarbonate (PC) membranes according to the invention, treated or not treated with heparin, ethylcellulose (EC) and hydroxypropylmethylcellulose (HPMC), to glucose (A), insulin (B) and IgGs (C) under static conditions.

[0143] FIG. 2: Insulin secretion by rat pancreatic islets stimulated with glucose through a PET membrane according to the invention, treated or not treated with heparin, EC and HPMC. A beginning of diffusion of the insulin starting from 4 hours and a permeability which appears to be improved at 24 hours by the surface treatment are observed.

[0144] FIG. 3: Images of the sections prepared 30 days after the implantation of poly(ethylene terephthalate) (PET) or polycarbonate (PC) membranes according to the invention, treated or not treated with heparin, EC and HPMC. The surface treatment decreases fibrosis and cell infiltration (black arrows) and increases vascularization (*) for the two types of membrane.

[0145] FIG. 4: Appearance of the bioartificial organs after 15 days of implantation in pigs. One of the devices is composed of monolayer PC membranes and the other of multilayer PET membranes. The device with PC membranes shows wide tears. The device with multilayer PET membranes does not, for its part, show any macroscopic damage. Said multilayer PET membranes were thus analyzed by scanning electron microscopy, which demonstrated no microcracks.

EXAMPLES

Example 1: Manufacture of Semi-Permeable Membranes

[0146] The membranes are manufactured such that two porous PET (poly(ethylene terephthalate)) layers were prepared from biocompatible PET films by the track-etching process, followed by lamination with the layer of non-woven PET having a density between 30 and 60 g/m2 (situated between the two porous biocompatible PET layers). A thermal lamination is carried out without the use of adhesives. One of the porous PET layers has a pore density between 2.Math.10.sup.9 and 7.Math.10.sup.9 pores/cm.sup.2 with an internal pore diameter between 10 and 30 nm. The thickness of this membrane is between 8 and 12 m. The other porous PET layer has a pore density between 10.sup.7 and 5.Math.10.sup.7 pores/cm.sup.2 with an internal pore diameter between 400 and 600 nm. The thickness of this membrane is between 30 and 50 m. The total thickness of the membrane is less than 200 m.

Example 2: Surface Treatment of the Membranes

[0147] The membranes prepared according to Example 1 were subjected to a surface treatment according to the protocol of Example 1 of WO 2012/017337.

[0148] The membranes are functionalized with a first layer of heparin mixed with a solution of ethylcellulose (EC), then covered with a layer of hydroxypropylmethyl-cellulose (HPMC).

Example 3: Characterization of the Membrane Permeability

[0149] Tests for glucose-permeability, insulin-permeability and immunoglobulin (IgG)-permeability of the previously prepared membranes were carried out according to the following protocol:

Material

[0150] Diffusion chamber consisting of a top compartment and a bottom compartment separated by the membrane, the permeability of which it is desired to test (the leaktightness between the two compartments is provided by a seal), glucose (Fischer Scientific, Illkirch, France, ref: G/0500/53), NaCl, IgG (Sigma, Lyon, France, ref: 19640), insulin (Sigma, ref: 19278), distilled water.

Preparation of Solutions

[0151] Physiological saline
For 1 l: 9 g of NaCl are dissolved in 1 l of distilled water. [0152] Glucose (4 g/1)
For 1 l: 4 g of glucose are dissolved in 1 l of physiological saline. [0153] IgG (5.75 g/ml)
For 60 ml: 34.5 l of stock solution of IgG (10 mg/ml) are diluted in 59.966 ml of physiological saline. [0154] Insulin (100 g/ml)
For 60 ml: 60 l of stock solution of insulin (10 mg/ml) are diluted in 59.960 ml of physiological saline.

Protocol

[0155] 3 ml of physiological saline are introduced into the bottom compartment of the diffusion chamber, and the membrane, the permeability of which it is desired to test, is placed on the physiological saline while avoiding the presence of air bubbles. 3 ml of glucose solution are introduced into the top compartment, then the diffusion chamber is closed with parafilm and is incubated at 37 C.

[0156] At the end of the incubation time, 1 ml of the solution contained in the top compartment of the diffusion chamber is removed after gentle homogenization. The membrane is then removed and 1 ml of the solution of the bottom compartment is removed after homogenization.

[0157] Enzymatic assaying of the glucose is carried out using the Glucose RTU kit (BioMrieux, Craponne, France ref: 61 269). The insulin and the IgGs are assayed using the bicinchonic acid (BCA) method by means of the Quantipro BCA Assay kit (Sigma, ref: QPBCA-1KT). The results are expressed as percentage permeability, calculated in the following way:

Permeability(as %)=(C.sub.bottom compartment/C.sub.top compartment+C.sub.bottom compartment)100

[0158] C: concentration of glucose, IgG or insulin.

[0159] At equilibrium, the concentrations in the top compartment and in the bottom compartment are identical, which corresponds to a maximum permeability of 50%.

Results

[0160] The results are shown in FIG. 1. Multilayer poly(ethylene terephthalate) (PET) membranes according to the invention (Example 1), and also prior art membranes as described in WO 02/060409 or WO 2012/017337, made of polycarbonate and having a layer of heparin mixed with EC and a layer of HPMC, were tested.

[0161] A slower diffusion of insulin and of glucose was observed with the PET membranes. Without wishing to be bound by this theory, it is possible that this is due to the presence of the multilayers of which they are composed.

[0162] The PET membranes are totally impermeable to IgGs.

Example 4: Semi-Permeable Membrane Implantation Tests

[0163] The membranes are implanted in the peritoneal cavity of healthy Wistar rats, according to the protocol described in Example 3 of WO 2012/017337.

[0164] The protocol relating to the taking of the samples was however modified and the samples are taken in the following way:

Taking Tissue Samples

[0165] Solutions Used [0166] 2.5% glutaraldehyde prepared, under a hood, from 25% glutaraldehyde (Sigma, ref: G58821010 ml) diluted to ten-fold in ultrapure water. [0167] PBS (reference: Gibco14190-094). [0168] Pot prefilled with 4% paraformaldehyde (Labonord, ref: PFFOR0060AF59001).

[0169] The membranes tested are the PET membranes according to the invention (multilayer) and the PC membranes of the prior art, optionally having undergone a surface treatment in order to deposit heparin, EC and HPMC.

[0170] The results are shown in FIG. 3: it is observed that the surface treatment with heparin reduces fibrosis and cell infiltration (black arrows) and increases vascularization (*) for the two types of membrane.

Example 5: Test for Glucose-Stimulation of Islets Through the Membrane

[0171] a) Isolation of Rat Pancreatic Islets

Animals Used

[0172] The animals used are male Wistar rats weighing 250-300 g (Janvier Laboratory, Le Genes St. Ile, France). The rats are housed in standard collective cages at a temperature of 231 C., and a hygrometry of 553% and with a cycle of 12 h of light and 12 h in the dark. SAFE-A04 feed (Villemoisson-sur-Orge, France) and water are available ad libitum. The animal experiments are carried out in accordance with European directive 2010/63/EU.

Removal of the Pancreas

[0173] The animal is anaesthetized with a mixture of Imalgene 1000 (active ingredient: ketamine, Centravet ref: IMA004) supplemented with 2.7 ml of Rompun (active ingredient: xylazin at 2%, Centravet ref: ROM001) injected intraperitoneally at a dose of 100 l/100 g of body weight.

[0174] After having verified the absence of reflexes of the animal, the latter is laid on its back. A laparotomy is then performed and the bile duct is ligatured at its duodenal opening. It is then catheterized at its hepatic opening and the animal is sacrificed by exsanguination. 10 ml of collagenase type XI (Sigma, ref: C7657) at 1 mg/ml at 4 C. are then injected into the pancreas by means of the catheter.

[0175] The pancreas is then removed and placed in a 50 ml Falcon tube containing 3.75 ml of sterile perfusion solution. This solution is composed of 500 ml of HBSS (Hanks Balanced Salt Solution, Lonza, ref: BE10-527F), 2.1 ml of 8.4% sodium bicarbonate, 1.175 ml of 1M calcium chloride and 12.5 ml of 1M HEPES. In order to limit the action of the enzyme during the removal, the tubes containing the pancreases are kept in ice.

Digestion

[0176] Immediately after the pancreases have been removed, the tube is placed in a waterbath at 37 C. for 10 minutes. It is then vigorously stirred for a few seconds in order for the tissue to be well dissociated. It is then made up with a cold washing solution. The washing solution is composed of M199 (Sigma, ref: M0393-50L) supplemented with 0.35 g/l of sodium bicarbonate (Sigma, ref: S-5761), with 10% of foetal calf serum (FCS, Lonza, ref: DE14-801F) and with 1% of anti-mycotic antibiotic (AMAB, Fisher, ref: W3473M).

[0177] The content of the tube is filtered on inserts (Corning Netwell inserts, Sigma, ref: CLS3480) and the filtrate is transferred into a 200 ml Corning tube which is centrifuged for 1 minute at 1200 rpm at 4 C. The supernatant is then removed and the pellet is resuspended with cold washing solution, then transferred into a 50 ml Falcon tube. After centrifugation for 1 minute at 1200 rpm at 4 C., a maximum amount of supernatant is removed before going on to the purification step.

Purification

[0178] The purification of the islets is carried out using a discontinuous gradient of Ficoll (Fisher, ref: BP525-500) which is composed of 3 solutions of different densities prepared in the laboratory: 1.108 (Ficoll 1): 1.108, 1.096 (Ficoll 2): 1.096 and 1.069 (Ficoll 3): 1.069.

[0179] The cell pellet is resuspended in 12 ml of Ficoll 1, and 10 ml of Ficoll 2 then of Ficoll 3 are carefully added on the top. Finally, 5 ml of PBS (Fisher, ref: 20012-019) are deposited on the Ficoll 3. The whole assembly is centrifuged for 4 minutes at 400 rpm at 4 C. and then for 12 minutes at 2000 rpm at 4 C. The braking and accelerating speeds of the centrifuge are adjusted to the minimum so as not to disturb the gradients.

[0180] The islets are recovered at the interface between the Ficoll 2 and the Ficoll 3, and are then washed three times in a cold washing solution in order to remove any trace of Ficoll.

Culturing

[0181] The islets are cultured in M199 medium (Gibco, ref: 23340-020) containing 10% of FCS (Lonza, ref: DE14-801F) and 1% of AMAB (Fisher, ref: W3473M) in untreated 25 cm2 flasks (Dutscher, ref: 690195), for 24 hours at 37 C. and in a humid atmosphere at 5% CO2.

[0182] b) Stimulation Test

[0183] Ten rat islets are placed in inserts (type of cylinders) at one end of which the PET membrane is attached. This membrane is oriented in such a way that the nanoporous membrane (which has pores with an internal diameter between 10 and 50 nm and which is selective for molecules up to 150 kDa) is on the inside of the insert, in contact with the rat islets, the layer which has the pores with a diameter of between 400 and 600 nm being oriented towards the outside of the insert.

[0184] The insert contains 400 l of Krebs solution containing 10% of FCS and 2.5 mM of glucose. The inserts thus filled are placed in wells of a 24-well plate containing 1 ml of Krebs solution containing 10% of FCS and 25 mM of glucose. The 24-well plate is then incubated at 37 C. and samples of medium contained in the wells are taken at 1 h, 2 h, 4 h, 6 h, 8 h and 24 h. The insulin is then assayed in the samples using the ELISA method (Mercodia, ref: 1250-01).

[0185] The islets are also sampled and placed in 50 l of lysis buffer (ThermoScientific, ref: 78501), supplemented with a protease inhibitor (ThermoScientific, ref: 78441), in order to extract the total proteins. The extraction is carried out by placing the tubes on ice for 30 min, while regularly vortexing the samples. The total protein content of the islets is determined by means of a Bradford assay and is used to normalize the secretion of insulin between the various islet cultures.

Example 7: Implantation and Explanation of MAILPAN in Pigs

[0186] An encapsulating chamber (MAILPAN, for MAcro-encapsulation d'ILots PANcratiques [macro-encapsulation of pancreatic islets]) is prepared according to the method described in WO 2012/010767. Two semi-permeable membranes are welded together. This encapsulating chamber has an internal sheet, and also connectors.

Anaesthesia

[0187] Premedication is systematic before any anesthesia and consists of the intramuscular administration of a combination of a butyrophenone: 2 mg/kg azaperone (Stresnil*) and of 10 mg/kg ketamine (Imalgene*).

[0188] General anesthesia is carried out according to the protocol described hereinafter: [0189] the animals are taken, premedicated, to the operating block and placed on the operating table lying on their side. [0190] A peripheral vein is catheterized (G 22) on one ear and its permeability is ensured by rinsing with a 0.9% NaCl solution. [0191] The induction is carried out by intravenous injection of a hypnotic (5 mg/kg thiopental or 4 mg/kg propofol) and of a curarising agent (0.1 mg/kg pancuronium). It is immediately followed by orotracheal intubation (Portex Blue Line, low-pressure balloon, calibre 6 for a subject weighing 25 to 35 kg) and by pulmonary ventilation using a semi-closed circular system connected to a respirator operating in controlled pressure mode. The ventilation (FiO.sub.2=0.5 FiN.sub.2O=0.5) is adjusted so as to maintain E.sub.CO.sub.2 between 35 and 45 mmHg. The respirator is a latest-generation human apparatus (GE Avance*, Aisys* or Aespire*) fitted with current flow rate, pressure and volume controls. [0192] The anaesthesia is maintained on inhalation mode with isoflurane (fraction inspired=2 vol %) with a fresh gas flow rate of 2 l/min of a 50%/50% O.sub.2N.sub.2O mixture serving as vector gas. [0193] If it proves to be necessary, the administration of subsequent doses of pancuronium provides optimum muscle relaxation under coverage of deep inhalation anaesthesia (MAC of isoflurane in pure O.sub.2=1.15 vol % and MAC of N.sub.2O=110 vol %.

MAILPAN Implantation

[0194] After anesthesia of the animals, the abdomen of the animals sent to sleep is made aseptic using 70% ethanol and then betadine (taking care not to cause hypothermia) and is shaved using a scalpel blade. A longitudinal incision of approximately 10 to 15 centimeters of the skin and muscle planes as far as the peritoneum is made in the middle of the cleared zone. After a median laparotomy, the prototype is implanted extraperitoneally, after being filled with physiological saline, and is attached to the wall with thread (Vicryl 2/0). The two catheters of the MAILPAN (one being used for the filling and the other for the emptying of the islets in the MAILPAN, in a period subsequent to the implantation) are connected to two injection chambers placed subcutaneously, before ligature of the peritoneum by sinusoidal movement, using 4-0 suture thread.

[0195] At the end of the surgery, the wounds are infiltrated with Naropein, and fentanyl will be administered IV before the animal is woken up. Fentanyl granules are administered per-operatively with the food intake, in a proportion of 2 mg/kg.

MAILPAN Explanation

[0196] The MAILPAN devices are explanted 15 days and 60 days post-transplantation under general anesthesia in order to evaluate the mechanical strength of the MAILPAN, the sterility thereof and the biocompatibility thereof (vascularization at the surface, absence of inflammation, absence of fibrosis and of inflammation on the surrounding tissues). Thus, samples of tissues surrounding the MAILPAN are taken at each explanation of the device for subsequent histological tests. The pigs are sacrificed after each explanation by intravenous injection of KCl.

[0197] The tissue samples are taken under the same conditions as for the rat (see Example 5: same solutions for tissues and membranes, same analyses carried out).

Example 8: Analyses of the Membranes by Scanning Electron Microscopy

[0198] After sampling, the membranes are rinsed in ultrapure water and fixed for 24 to 48 h at 4 C. in glutaraldehyde (Sigma, ref: G5882) diluted to 2.5%. The fixed membranes are then rinsed for 10 minutes in ultrapure water.

[0199] The samples are then dehydrated using successive baths of ethanol: two baths of 10 minutes in 50% ethanol, one bath of 25 minutes in 70% ethanol, then one bath of 10 minutes in 95% ethanol and, finally, two baths of 10 minutes in 100% ethanol. In order to completely remove the traces of water which might still be present in the samples, an incubation for 2 minutes is carried out in hexamethyldisilazane (HMDS) (Sigma, ref: 440191).

[0200] After drying in the open air, the samples are then adhesively bonded flat on blocks (Delta Microscopies, ref: 75220), using carbon-conducting adhesive (Delta Microscopies, ref: 76510).

[0201] Once the adhesive has solidified, the samples are metallized by depositing a thin layer of gold-palladium, and then of carbon.

[0202] The observation is carried out on a field-effect scanning electron microscope (SEM) (Hitachi S800) (in vitro imaging platform of the Neurochemistry Centre of Strasbourg) at a voltage of 5 KV, which makes it possible to obtain good resolution without damaging the samples.

Results

[0203] It is observed that the device produced with PC membranes shows wide tears (FIG. 4).

[0204] On the other hand, the device produced with the multilayer PET membranes does not, for its part, show any macroscopic damage. Said membranes were thus analyzed by scanning electron microscopy, which demonstrated no microcracks (FIG. 4).

[0205] It therefore appears that the membranes according to the invention allow a diffusion similar to that observed for the prior art membranes and clearly have the property of semi-permeability (blocking IgGs, and other proteins of the immune system). These membranes exhibit much better resistance when they are used in a bioartificial organ implanted in vivo.

[0206] Further data has been obtained for tensile strength in vitro, for PET membranes. The strength is slightly higher for a tri-layer membrane (two porous PET membranes surrounding a non-woven PET membrane), than for a two-layer membrane (one porous PET membrane laminated on a non-woven PET membrane). The tensile strength of the two-layer membrane is higher than the one for a mono-layer porous PET membrane.