JP2007195573A - Method for separating pancreatic island for transplant - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for separating a pancreatic island for a transplant. <P>SOLUTION: The method for separating a pancreatic island for a transplant increases the dissolved oxygen concentration in a pancreatic island separating circuit directly from outside. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a method for isolating islets for transplantation.

  Since the advent of the Edmonton protocol, islet transplantation has attracted attention all over the world and is now being established as an ideal radical treatment for type 1 diabetes (Non-patent Document 1). In Japan, pancreatic islet transplantation was started in April 2004. In Japan, however, there are extremely few brain death donors due to social and religious reasons, and the source of the pancreas for pancreatic islet transplantation is important. The current situation is that we have to rely on stopped donors. The biggest problem with cardiac arrest donors as a source of pancreas is that the physiological activity of the islets is reduced due to the longer warm ischemic time compared to brain-dead donors. Therefore, it was generally considered that islet transplantation from a cardiac arrest donor was impossible, but recent results in Japan showed that it is sufficiently possible to shorten the warm ischemic time (Non-patent Document 2). ).

  According to the data so far, it is known that the quality and quantity of the islets obtained are both extremely reduced when the warm ischemic time exceeds 30 minutes. In Japan, it is often difficult to shorten the warm ischemic time in islet transplantation from a cardiac arrest donor, that is, to remove the pancreas within 30 minutes after cardiac arrest. It has been desired to develop a technique capable of obtaining a quality and quantity of islets sufficient for islet transplantation even if the time exceeds 30 minutes.

  Furthermore, it has recently been reported that islet yield can be increased by a factor of 2 by using oxygenated PFC during rat pancreas digestion (Non-patent Document 3).

However, the following problems are known for PFC.
1. Because it is known that the PFC itself is toxic, it is not preferable to contact the islets for a long time, that is, when the islets are separated from the pancreas, they are much more fragile than if they are in the pancreas, Toxicity that is not a problem in the pancreas can be fatal.
2. The most important thing in islet transplantation is to efficiently decompose the pancreas and recover more islets. In the process, it is most important to maintain the activity of the enzyme, and injecting PFC into the circuit is likely to affect the activity, which is not realistic in view of clinical application.
3. Furthermore, PFC has an extremely high specific gravity and is hydrophobic, so that fusion with the enzyme solution is not observed in the circuit, preventing the maximum effect of the enzyme. Rather, it is expected that the digestion time will be prolonged, and even if the yield increases, the resulting islet will be subject to a lot of stress and will eventually lead to “quality degradation” of the islet. A short digestion time is known to reduce the stress on the islets and also reduce the release of inflammatory cytokines, leading to an improvement in the “quality” of the islets (Non-Patent Document 4).

  Therefore, development of an islet isolation method that does not have the above-described problems has been desired.

James Shapiro et al., N Engl J Med, 343 (4): 230, 2000 Matsumoto Shinichi et al., 32nd Pancreatic / Islet Transplantation Society: C-12, 2005 Kuroda, Kobe University COE Report, 25-27, 2005 Masafumi Goto et al., Transplantation: 78 (9): 1367, 2004

  An object of the present invention is to provide a method for separating islets for transplantation.

  As a result of continual research to solve the above problems, the present inventors have found that by increasing the dissolved oxygen concentration in the islet separation circuit, it is possible to obtain islets of sufficient quality and quantity for islet transplantation. The present invention has been completed.

That is, the present invention relates to the following.
1. A method for separating islets for transplantation, comprising a step of digesting the pancreas with an enzyme in the islet separation circuit, wherein the dissolved oxygen concentration in the islet separation circuit is increased externally and directly, ,
2. The method for separating islets for transplantation according to 1 above, wherein the dissolved oxygen concentration in the islet separation circuit is 30 to 38 μg / ml,
3. 3. The method for separating transplant islets according to 1 or 2 above, wherein the islets are derived from pigs or humans,
4). The method for isolating a pancreatic islet according to 3 above, wherein the islet is derived from a single individual of pig or human,
5). A pancreatic islet obtained by using the separation method according to any one of 1 to 4 above.

  In the present invention, “islet” is a cell mass composed of an average of about 2000 islet cells. Since this cell mass itself has blood glucose sensing ability, it is desirable that the cell mass is in the state of islets instead of islet cells when used for transplantation.

  In the present invention, the “islet separation circuit” is a perfusion circuit containing an enzyme solution for extracting islets from the pancreas. Among them, Ricordi Chamber (Ricordi C, Diabetes: 38: suppl 1: 140: 1989) in which the pancreas is placed, a warmer to maintain the enzyme's optimal activity temperature of 37 ° C, and a circuit are perfused A peristaltic pump or the like (see FIG. 1).

  In the present invention, “to increase the dissolved oxygen concentration externally and directly” means that islet separation is performed by applying oxygen from the outside of the islet separation circuit to the islet separation circuit without passing through an oxygen transport medium, for example, PFC. To increase the dissolved oxygen concentration in the circuit.

  In the present invention, “islet for transplantation” refers to an islet having a function sufficient to restore the pathological state of type 1 diabetes when transplanted to a mammal such as a human. This function can be confirmed, for example, by an in vivo islet function test.

  Among the many islet function tests, the most reliable one is the so-called “in vivo islet function test”, which confirms whether animals that have actually become diabetic can be cured with the islets. Has been known for a long time. Despite the well-known fact that this test best reflects actual islet function, less clinically used, this test requires a larger amount of islets than in vitro tests. This is because it has a drawback that it takes several days until the result of transplantation is clearly understood.

  All recipients used for transplantation can avoid immune responses if allogeneic, so the results will all depend on islet function. However, in the case of porcine islet isolation, it is practically impossible to prepare similar siblings, and even if recent genetic techniques are used, a considerable amount of money is required. Therefore, in the case of pigs, the function of the islets is evaluated by transplanting into nude mice that can exclude the effects of immune responses (those that lack T cells because they do not have thymus and do not cause immune responses). It is carried out. This method has been established since ancient times and has been incorporated in many papers (by Rayat GR, Transplantation: 69 (6): 1084: 2000).

  The method of the present invention can prepare a transplant that can be used for islet transplantation, which is an ideal radical treatment for type 1 diabetes.

  The means for externally and directly increasing the dissolved oxygen concentration for the method for separating islets for transplantation of the present invention is not particularly limited as long as it is a conventionally known means, but preferably from an oxygen storage device such as an oxygen cylinder. This is an air injection method in which oxygen is directly injected into the circuit. For speeding up, the purity of oxygen is preferably 100%, and the injection pressure is preferably 1 L / min of oxygen.

  The increased dissolved oxygen concentration of the present invention is preferably 30-38 μg / ml, more preferably 35-38 μg / ml, most preferably 38 μg / ml.

  The method for measuring the dissolved oxygen concentration in the present invention is not particularly limited as long as it is a conventionally known method. For example, the dissolved oxygen concentration is measured in a circuit solution by a dissolved oxygen meter toughness (Sato Corporation).

  The method for separating the islets for transplantation in the present invention is not particularly limited as long as it is a conventionally known method, but is preferably called the Ricordi modified method, in which the pancreas filled with the enzyme is placed in a Ricordi chamber, This is a method in which the pancreatic pancreas is chemically and physically digested by perfusing a pancreatic islet separation solution that has been heated to 37 ° C. with a peristaltic pump, and only the islet that is the final target product is extracted (Goto). (See Masafumi et al., Transplantation: 78 (9): 1367, 2004).

  The source of pancreatic islets for transplantation in the present invention is a mammal, preferably a human, but can also include pigs when performing xenogeneic islet transplantation. Also, this source is preferably derived from a single individual.

  The present invention also relates to a pancreatic islet obtained by using the separation method of the present invention.

  As a model of islet transplantation from clinical cardiac arrest donors, we performed islet isolation and transplantation experiments from cardiac arrest pigs that received 40 minutes of warm ischemia. Pigs are said to be one of the most difficult mammals in which islets are extremely fragile and isolated, but because the pancreas size and the amino acid sequence of insulin are very similar to humans, they become good models, Pigs were selected as model animals.

  In the conventional separation method, the yield of islets is extremely low in the porcine model and the “quality” of the islets is also reduced, so that transplanting the islets of the product can cure diabetic mice. There wasn't. On the other hand, when the insufflation method was performed, diabetic mice could be cured, and in vitro results proved that the quality of the product islet was improved.

  Originally, an insufflation method was introduced with the aim of improving the quality of the islets, so far it was within the expected range, but surprisingly, it has been found that the yield of the islets itself increases significantly. This is probably because the islets were damaged due to stress due to long warm ischemia and caused necrosis, but the number of surviving islets was increased by the introduction of oxygenation by the insufflation method.

  In principle, oxygen injection was performed continuously. As shown in FIG. 2, the maximum volume of 38 μg / ml is reached in about 5 minutes after the start of continuous oxygen injection. Thereafter, when the bubbling is stopped, the dissolved oxygen concentration quickly decreases to 18 μg / ml, and the concentration is maintained for at least 15 minutes.

  Therefore, when it is applied to an actual islet isolation case, it usually takes about 20 minutes until the digestion stop process, so that the target dissolved oxygen concentration of 38 μg / ml was sufficiently maintained by continuous oxygen injection. Next, after the digestion was stopped, in order to collect the pancreatic degradation product by the enzyme, washing was started with a new 1 L drip bottle solution at a rate of 200 ml / min. Since the dissolved oxygen concentration does not reach the maximum volume of 38 μg / ml within 5 minutes of using up the solution, the infusion bottle was pre-bubbled in order to make the dissolved oxygen concentration the maximum volume in advance.

  By doing so, it was possible to quickly maintain the maximum capacity of 38 μg / ml even after changing the infusion bottle. Since about 7L of washing solution is usually required, instead of using an infusion bottle, a method of bubbling seven solutions in a large beaker could be considered, but in this case there is no closed circuit, so contamination This is not a good option when considering the clinical application.

  Grafted immunostained image: When creating a model diabetic mouse, a toxic substance called STZ is usually injected to selectively destroy the β cells of the islets, creating a pathology as if it were a type 1 diabetic patient. Normally, when STZ is injected at 250 mg / kg from the tail vein, almost 100% of mice develop diabetes, but very rarely, they are resistant to STZ and some of their β cells remain. It is known that mice can be seen. In such a case, it is unclear whether the normalization of blood glucose level after transplantation is due to grafting or the recovery of own β cells. Therefore, in such a case, the graft is removed and the mouse is observed again to evaluate whether it returns to diabetes. At that time, it is very important to carry out insulin immunohistochemical staining of the graft to confirm whether or not the graft actually has insulin, and this is an established graft evaluation method.

  The graft of the present invention has abundant insulin even after about 2 months after transplantation, and it was confirmed that the graft was clearly good.

Dissolved oxygen concentration in the circuit by insufflation method The dissolved oxygen meter cannot measure accurately when the solution is stationary, and requires a water surface of at least 6 cm, so it is placed directly in the infusion bottle and closed again. In order to maintain the circuit state, the dissolved oxygen meter injection portion was tightly sealed with gummed tape. The islet separation circuit and the islet separation solution were reproduced exactly the same as those used for normal porcine islet separation. The results are as shown in FIG.

Islet yield from pigs with approximately 40 minutes of warm ischemia Porcine pancreas was sent to the Sendai Meat Market Center with permission from Sendai City and separated directly from other internal organs. After the cardiac arrest of the pig, pancreas extraction was started from 25 minutes, trimming was performed after extraction, and only the pancreatic tail was immersed in a cooled UW solution and transported. In the laboratory, a pancreatic digestive enzyme called 500 mg liberase was injected from the main pancreatic duct, and then the fat, blood vessels, and membrane tissue around the pancreas were trimmed. After sufficient trimming, the pancreas was cut into 6 pieces and placed in the Ricordi chamber in the islet separation circuit. The islet isolation circuit was perfused at a rate of 200 ml / min using a peristaltic pump to chemically and physically promote pancreatic digestion. The sample was taken out from the circuit over time, and the digestive condition of the pancreas was confirmed. When the islets were sufficiently released from other tissues, the digestion in the circuit was started to be collected. Since the collected cell pellet also contains exocrine cells, specific gravity separation was performed using COBE2991 which is a blood cell separator. Finally, a fraction containing a large amount of islets was selected, and the yield was measured under a microscope, mixed with the culture solution, and cultured in a 37 ° incubator. The results are as shown in FIG.

Glucose-loaded islet function test Sixty islets with a diameter of 150 μm were randomly selected and distributed into three tubes containing 1.67 mM low-concentration glucose. After incubating at 37 degrees for 30 minutes, the supernatant was recovered, and then incubated with 16.7 mM high-concentration glucose for 30 minutes, and the supernatant was recovered in the same manner. The insulin concentration in each supernatant was measured by ELISA (Mercodia), and the value obtained by dividing the amount of insulin released in the high concentration glucose solution by the amount of insulin released in the low concentration glucose solution was called the Stimulation Index. Used for evaluation. The results are as shown in FIG.

Islet transplantation for diabetic athymic mice Athymic mice were previously diabetic with 250 mg / kg STZ. Approximately 3000 IEQs of porcine islets were transplanted under the kidney capsule of the mouse, and the blood glucose state of the mouse was observed and used for the evaluation of the graft. After 8 weeks, the kidney containing the graft was removed and the effect of the graft was reconfirmed. The results are as shown in FIG.

FIG. 1 is an islet separation circuit. FIG. 2 shows the dissolved oxygen concentration in the circuit by the air injection method. The vertical axis represents the oxygen concentration (μg / ml). FIG. 3 is an islet yield from a pig about 40 minutes warm ischemia. API is an abbreviation for Adult Pig Islets. IEQs is an abbreviation of Islet Equivalents (islet equivalent), and is an international unit indicating the volume of islets, defining an islet with a diameter of 150 μm as 1. Therefore, the vertical axis of the graph indicates the amount of islets obtained from 1 g of pancreas weight. FIG. 4 is a glucose tolerance islet function test. The vertical axis represents the Stimulation Index (glucose stimulation index). FIG. 5 shows the results of islet transplantation for diabetic athymic mice. The vertical axis represents blood glucose concentration (mg / dl), and the horizontal axis represents the number of days after transplantation.

Claims (5)

  A method for separating islets for transplantation, comprising a step of digesting the pancreas with an enzyme in the islet separation circuit, wherein the dissolved oxygen concentration in the islet separation circuit is increased externally and directly, .   The method for separating islets for transplantation according to claim 1, wherein the dissolved oxygen concentration in the islet separation circuit is 30 to 38 µg / ml.   The method for separating islets for transplantation according to claim 1 or 2, wherein the islets are derived from pigs or humans.   The method for separating islets for transplantation according to claim 3, wherein the islets are derived from a single individual of pig or human.   An islet for transplantation obtained by using the separation method according to any one of claims 1 to 4.