WO2019173992A1

WO2019173992A1 - Pancreatic islet separation device and method

Info

Publication number: WO2019173992A1
Application number: PCT/CN2018/079062
Authority: WO
Inventors: 杜文军
Original assignee: 深圳市云杉医疗管理有限公司
Priority date: 2018-03-14
Filing date: 2018-03-14
Publication date: 2019-09-19

Abstract

A pancreatic islet separation device and method. The device comprises a cavity (1) and a filter mechanism (2). The filter mechanism divides the inner cavity of the cavity (1) into a first chamber and a second chamber; the first chamber is provided with a first opening (111); the second chamber is provided with a second opening (112). According to the device, the cavity (1) is divided into two chambers by means of the filter mechanism (2); after a pancreas digests in one chamber, a released pancreatic islet passes through the filter mechanism (2) from the first chamber to enter the second chamber; the second opening (112) is intermittently opened by unscrewing to allow the pancreatic fluid in the second chamber to flow out, thereby avoiding excessive digestion of the pancreatic islet; the device is simple in structure, low in costs, and convenient to operate.

Description

Islet separation device and method

Technical field

The invention relates to the field of biomedical technology, and in particular to an islet separating device and method.

Background technique

The use of islet cell transplantation for the treatment of diabetes, as well as the application of islet cells for basic biological and pharmaceutical research, involves the isolation of functional islet cells from pancreatic tissue. The pancreas is composed of endocrine glands (also known as Langerhans cells) and exocrine glands, which are scattered in the acinus and account for only 1-2% of the total pancreas.

At present, the basic technical principle of islet cells isolated from pancreatic tissue is to digest pancreatic connective tissue with collagenase, release islet cells, and separate islet cells and exocrine cells by subsequent purification steps [Ricordi C. et al., Isolation of the elusive pig islet.Surgery.1990.107:688-694]. Such a method principle can be applied to small animals (eg, rat [CN1699556A]), large animals (eg, pig [CN101033460A]), and human islet cells [semi-automatic digestion and purification of human islet cells. Experimental Journal of Southern Medical University .2007.27(6): 824-826] Separation.

Of course, due to the differences in composition and structure of pancreas of different species, the specific separation process requires targeted optimization to establish a suitable method to stably obtain higher quantities and quality of islets. In addition, although the islet separation principle is simple, the process involves enzymes, enzyme digestion enzyme solution concentration and pH, digestive enzyme solution perfusion process, digestion time, termination of digestion and washing process, etc., leading to islets The standardization of separation methods is difficult. Many times, it depends on the experience of the operators. There are still many places to be further optimized.

For small animals, because the pancreas is small, the digestion process is relatively uniform, relatively fast, and the general laboratory equipment can be satisfied, such as centrifuge tubes (50 ml), sieves, constant temperature water bath equipment and centrifuges. For the separation of large animals (mainly pigs) and human islets, due to the large size of the pancreas, it is difficult to achieve uniform digestion by completely artificial methods, and it is difficult to ensure the quantity and quality of islets harvested.

Semi-automated equipment has been developed abroad for islet isolation. The most important representative is the Ricordi islet isolation system from Biorep, USA (the main function is pancreatic digestion), combined with COBE 2991 automatic islet cell purification equipment from Terumo BCT, USA. For large-scale, standardized islet preparation. However, these devices are very expensive, generally unacceptable in the laboratory, and have some drawbacks. The islet separation process is complicated. For example, in the Ricordi system, it is generally sampled from a circulating digestive enzyme solution to observe the presence or absence of islet cells and exocrine cells to determine the digestive state of the pancreatic tissue in the digestive cavity to determine whether to continue digestion or to terminate digestion. However, in practice, operations are difficult and require very skilled technology.

Researchers have also developed corresponding systems for islet isolation in large animals and humans [Experimental study on separation and purification of human islet cells by semi-automatic digestion. Journal of Southern Medical University. 2007.27(6): 824-826; CN205556072U; CN102304477A; CN103952371A; CN205741019U], but basically a simple copy of Ricordi's system, integrated cycle digestion and temperature control function, and does not solve the key problem of prolonged digestion during the process of excessive digestion of islets, thereby reducing islet yield. In the islet isolation method, the large animal pancreas is digested due to its large volume, so that the digestion time is longer, and the release of islets during the digestion process is gradual. We have found that as long as the segmentation digestion is achieved during the digestion process, the excessive digestion of islets can be effectively controlled, thereby significantly increasing the yield and quality of islets.

Summary of the invention

In view of the deficiencies of the prior art, the present invention provides an islet separation device and method for isolating a cavity into two chambers by a filtering mechanism. After the pancreas is digested in one end chamber, the islet is released from the first cavity. The chamber is filtered to the second chamber by the filtering mechanism, and the second opening can be opened intermittently to release the islet liquid in the second chamber to avoid excessive digestion of the islets; and the existing islet separating device has the advantages of complicated structure and high cost. Inconvenient operation.

In order to achieve the above object, the technical solution of the present invention is as follows:

An islet separating device for digesting a pancreas, including a cavity and a filtering mechanism. The filtering mechanism isolates the inner cavity of the cavity into a first chamber and a second chamber. The first chamber is provided with a first opening. The second chamber is provided with a second opening.

Further, the cavity comprises a first spherical cap cavity and a second spherical cap cavity. The filtering mechanism connects the first spherical cap cavity with the second spherical cap cavity; and the filtering mechanism isolates the inner cavity of the first spherical cap cavity from the inner cavity of the second spherical cap cavity, The first chamber and the second chamber are formed. The first opening is disposed on the first spherical cap cavity. The second opening is disposed on the second spherical cavity.

Further, the first opening and the second opening respectively have a cover. The cover is used to seal the cavity.

Further, the filtering mechanism comprises a cavity connecting barrel and a screen. The diameter of the bottom surface of the first spherical cap cavity and the second spherical cap cavity is the same as the diameter of the screen. The screen is installed in the cavity connecting barrel, and the internal space of the cavity connecting barrel is divided into two parts. One end of the cavity connecting barrel is sleeved on the first spherical cap cavity, and the other end of the cavity connecting barrel is sleeved on the second spherical cap cavity.

Further, the screen has a disk shape. A plurality of circular filtering through holes are provided on the screen surface of the screen.

Further, opposite sides of the side wall of the cavity connecting barrel are respectively provided with opposite external threads. On the first spherical cap cavity, an internal thread is provided at the junction with the cavity connecting barrel. On the second spherical cap cavity, an internal thread is provided at the junction with the cavity connecting barrel. The first spherical cap cavity and the second spherical cap cavity are respectively engaged with the cavity connecting barrel by threads.

Further, external threads are respectively disposed on the first opening and the second opening. The cover of the first opening is provided with an internal thread. The cover of the second opening is provided with an internal thread. The cover of the first opening is screwed onto the first opening. The cover of the second opening is screwed onto the second opening.

Further, the cavity is made of a transparent material.

Further, the cavity and the cavity connecting barrel are made of a plastic material. The screen is made of steel.

Further, the volume ratio of the first chamber and the second chamber is between 1:1 and 10:1.

Further, the axis of the first opening coincides with the axis of the second opening.

An islet isolation method comprising the following steps:

S1: obtaining a pancreas of a fresh animal;

S2: Elimination of connective tissue on the pancreas;

S3: configuring a digestive enzyme solution;

S4: placing the pancreas in a petri dish and placing it on an ice bath; using a syringe to remove the enzyme solution, and injecting the digestive enzyme solution into each acinar structure of the pancreas; after the acinus is filled with the digestive enzyme solution, the pancreas is filled Divided into small pieces of tissue;

S5: covering the lid of the first opening on the islet separating device; placing the pancreatic tissue divided into small pieces from the second opening into the first chamber of the islet separating device, covering the lid of the second opening on the islet separating device; and islet The separation device was incubated in a 37° water bath to digest the pancreatic tissue; start timing, and gently shake the islet separation device for 8-15 minutes; the filtration mechanism has a filter through hole diameter of 450 μm; open the lid on the second opening, The filtrate was collected; the filtrate was filtered through a 200 μm pore size sieve to collect the filtrate; the filtrate was centrifuged; the centrifuged cell pellet was taken out; the cell pellet was resuspended in the medium, and the sample was collected for the first time and placed in an ice bath. stand-by.

The beneficial effects of the invention:

(1) The device separates the cavity into two chambers by a filtering mechanism, and after the pancreas is digested in the chamber at one end, the released islets are filtered from the first chamber through the filtering mechanism to the second chamber, which can be intermittently rotated. The second opening is opened to release the islet liquid in the second chamber to avoid excessive digestion of the islets; and the structure is simple, the cost is low, and the operation is convenient.

(2) The device assembles and isolates the first spherical cap cavity and the second spherical cap cavity into two chambers through a filtering mechanism, and the assembly is simple and convenient to use.

DRAWINGS

Fig. 1 is a schematic perspective view showing the islet separation device of the present invention.

The reference numerals of FIG. 1 are: a cavity 1, a filter mechanism 2, a first spherical cavity 11, a second spherical cavity 12, a cavity connecting cylinder 21, a screen 22, and a first opening 111. The second opening 112 and the cover 10.

detailed description

The invention will now be further elucidated with reference to the drawings and embodiments.

As shown in Fig. 1, an islet separating device is used for digesting the pancreas to separate the islets, including the cavity 1, the filtering mechanism 2.

The filter mechanism 2 isolates the inner cavity of the cavity 1 into a first chamber and a second chamber. The first chamber is provided with a first opening 111 with a cover 10. The second chamber is provided with a second opening 112 with a cover 10.

The pancreas is placed in the first chamber, and the digestive enzyme solution is added from the first opening 111 to the first chamber. After the first opening 111 and the second opening 112 cover the cover 10, the cavity 1 is sealed; it is placed under a certain temperature condition, and the device is shaken (the device can be placed on the cover 10 and placed on the shaker function) In the water bath, set the temperature). After the pancreas has been digested for a certain period of time, the islets released by the pancreas, the digestive liquid containing the small cell mass are filtered from the first chamber through the filter mechanism 2 to the second chamber. In this way, the second opening 112 can be unscrewed intermittently, and the digested liquid containing the small cell mass in the second chamber can be released. The device has the advantages of simple structure, low cost and convenient operation. The digestive liquid can be filtered from the first chamber to the second chamber and released intermittently to avoid over-digestion of the islets; and the remaining pancreatic tissue is isolated by the filtering mechanism 2 in the first chamber to continue digestion; The second opening 112 releases the digestive liquid and realizes the segmental digestion of the pancreas, which can effectively control the excessive digestion of the islets, thereby significantly increasing the yield and quality of the islets.

The digestive enzyme solution was prepared according to the weight of pancreatic tissue, and a corresponding volume of 1.5 mg/ml collagenase V solution was prepared at a ratio of 5 ml/g of pancreatic tissue. After the digestive liquid was released, it was centrifuged, first centrifuged at 1700 r/min for 3 minutes, then centrifuged at 2400 r/min for 3 minutes, and the precipitates were collected separately. The cell pellet was taken, resuspended in the medium, and the sample was collected for the first time. Waiting in the ice bath.

Preferably, the axis of the first opening 111 coincides with the axis of the second opening 112.

Preferably, the volume ratio of the first chamber to the second chamber is between 1:1 and 10:1.

Specifically, the cavity 1 includes a first spherical cap cavity 11 and a second spherical cap cavity 12.

The first opening 111 is disposed on the first spherical cap cavity 11. The second opening 112 is disposed on the second spherical cap cavity 12. The filtering mechanism 2 connects the first spherical cavity 11 and the second spherical cavity 12, and isolates the inner cavity of the first spherical cavity 11 and the second spherical cavity 12; the filtering mechanism 2 The inner cavity of the first spherical cap cavity 11 and the inner cavity of the second spherical cap cavity 12 are separated into a first chamber and a second chamber.

Specifically, the filter mechanism 2 includes a cavity connecting cylinder 21 and a screen 22.

The diameter of the bottom surface of the first spherical cap cavity 11 and the second spherical cap cavity 12 is the same as the diameter of the screen 22. The screen 22 is installed in the cavity connecting cylinder 21, and the internal space of the cavity connecting cylinder 21 is cross-sectioned into two parts, left and right. One end of the cavity connecting cylinder 21 is sleeved or sleeved on the first spherical cap cavity 11, and the other end of the cavity connecting cylinder 21 is jacketed or sleeved on the second spherical cap cavity 12.

Preferably, the screen 22 is in the shape of a disk. A plurality of micron-sized circular or square filter through holes are provided on the disk surface of the screen 22. The circular filter through hole has a square filter through hole, which can improve the transmittance of the islet solution.

Preferably, opposite sides of the side wall of the cavity connecting cylinder 21 are respectively provided with opposite external threads. On the first spherical cap cavity 11, an internal thread is provided at the junction with the cavity connecting cylinder 21. On the second spherical cap cavity 12, an internal thread is provided at the junction with the cavity connecting cylinder 21. The first spherical cap cavity 11 and the second spherical cap cavity 12 are respectively engaged with the cavity connecting cylinder 21 by threads.

Preferably, the first opening 111 and the second opening 112 are respectively provided with external threads. The cover 10 of the first opening 111 is provided with internal threads. The cover 10 of the second opening 112 is provided with internal threads. The cover 10 of the first opening 111 is screwed onto the first opening 111. The cover 10 of the second opening 112 is screwed onto the second opening 112.

Preferably, the cavity 1 is made of a transparent plastic material. The cavity 1 is selected from medical grade hard transparent materials, including but not limited to PC, PSU, PS, K resin, PVC, and the like.

Preferably, the cover 10 is made of a plastic material. The cover 10 is selected from medical grade rigid plastic materials, including but not limited to ABS, PEEK, PTFE, PET, PBT, PPSU, PP, PC, PSU, PS, K resin, PVC, and the like.

Preferably, the cavity connecting cylinder 21 is made of a plastic material.

Preferably, the screen 22 is made of a steel material. The medical grade steel is selected, and the filter through hole of the screen 22 is a circular hole structure formed by laser drilling.

In addition, the present invention also discloses an islet isolation method, which comprises the above-mentioned islet separation device, comprising the following steps:

S1, pig pancreas: adult pigs (0.5-5 years old healthy pigs), bloodletting (artery, heart bleeding, by cutting the carotid artery with a knife or piercing the heart), after about 1-2 minutes, waiting for the pig to stop Breathing, immediately separate the whole pancreas from the surrounding tissue, remove the pancreas, wrap it in plastic wrap, store it in an ice bath, and quickly transport it back to the laboratory to prepare for islet isolation. Note: It is recommended to perform islet isolation immediately, but it can also be 4 Degree retention, separation within 3-4 hours does not significantly affect the separation effect);

S2. Eliminate connective tissue: In the ultra-clean platform, place the pancreatic tissue in a large petri dish, remove the fat, omentum, blood and other attached tissues with tweezers and surgical scissors, then weigh the net, take the whole pancreas or cut with a surgical scissors. Take the required weight of pancreatic tissue (for example, 20 grams) for digestion;

S3, preparing a digestive enzyme solution: according to the weight of the pancreas tissue, a corresponding volume of 1.5 mg / ml collagenase V solution is prepared according to the ratio of 5 ml / g pancreatic tissue;

S4, perfusion treatment: place the pancreas (or part of the pancreatic tissue) in a new culture dish, place it on an ice bath, and use a syringe (5-20 ml syringe is recommended, with a 1 ml syringe standard needle) to absorb collagenase V digestive enzymes. The solution is injected into each acinar structure, and the acinar membrane is inflated to fill the digestive enzyme solution, and then the pancreas is pulled into small pieces (3-5 mm size) with tweezers.

S5, pancreas digestion: first cover the cover 10 on the second opening 112, and assemble the second spherical cavity 12 and the filter mechanism 2; transfer the pancreatic tissue injected with the digestive enzyme solution to the first spherical crown Inside the cavity 11; cover the cover 10 on the first opening 111, incubate the islet separating device in a 37-degree water bath, digest the pancreatic tissue, start timing, and gently shake the device for 8-15 minutes ( Note: The incubation time varies slightly depending on the batch of the enzyme, the strain of the pig and the individual, and needs to be slightly adjusted to lengthen or shorten the digestion time); the filter through hole diameter of the filter mechanism 2 is 450 μm; the second opening 112 is opened. The cover 10 collects the filtrate (the tissue trapped on the sieve is left to be treated in the next step); the filtrate is filtered through a 200 μm pore size sieve, and the filtrate is collected (the tissue trapped on the sieve is left to be treated in the next step); Centrifuge at 1700r/min for 3 minutes, then centrifuge at 2400r/min for 3 minutes, collect the precipitate separately, take the cell pellet, resuspend in the medium, record the sample for the first time, and put it in the ice bath for use; (Note: 1st Secondary collection of centrifugation supernatant (ie enzyme Liquid), collected and used in the following steps).

The above is only a preferred embodiment of the present invention, and the present invention is not limited to the above embodiment. It is to be understood that other modifications and changes which are obvious to those skilled in the art without departing from the scope of the invention are intended to be included within the scope of the invention.

Claims

An islet isolation device for digesting a pancreas characterized by:

Including a cavity (1), a filter mechanism (2);

The filtering mechanism (2) isolates the inner cavity of the cavity (1) into a first chamber and a second chamber;

The first chamber is provided with a first opening (111);

The second chamber is provided with a second opening (112).
The islet isolation device according to claim 1, wherein:

The cavity (1) comprises a first spherical cap cavity (11) and a second spherical cap cavity (12);

The filtering mechanism (2) engages the first spherical cap cavity (11) with the second spherical cap cavity (12); and the filtering mechanism (2) places the first spherical cap cavity (11) The cavity is isolated from the inner cavity of the second spherical cavity (12) to form the first chamber and the second chamber;

The first opening (111) is disposed on the first spherical cavity (11);

The second opening (112) is disposed on the second spherical cavity (12).
The islet isolation device according to claim 1 or 2, wherein:

a first opening (111) and a second opening (112) respectively have a cover (10);

The cover (10) is used to seal the cavity (1).
The islet isolation device according to claim 2, wherein:

The filter mechanism (2) comprises a cavity connecting cylinder (21) and a screen (22);

The screen (22) is installed in the cavity connecting cylinder (21), and the internal space of the cavity connecting cylinder (21) is divided into two parts;

One end of the cavity connecting cylinder (21) is sleeved on the first spherical cap cavity (11), and the other end of the cavity connecting cylinder (21) is sleeved on the second spherical cap cavity (12).
The islet isolation device according to claim 4, wherein:

The screen (22) is in the shape of a disk;

A plurality of filter through holes are provided on the screen surface of the screen (22).
The islet isolation device according to claim 4, wherein:

Opposite external threads are respectively disposed on both sides of the side wall of the cavity connecting cylinder (21);

An internal thread is arranged on the first spherical cap cavity (11) at the junction with the cavity connecting cylinder (21);

The second spherical cap cavity (12) is provided with an internal thread at the junction with the cavity connecting cylinder (21);

The first spherical cap cavity (11) and the second spherical cap cavity (12) are respectively threadedly engaged with the cavity connecting cylinder (21).
The islet isolation device according to claim 3, wherein:

The first opening (111) and the second opening (112) are respectively provided with external threads;

The cover (10) of the first opening (111) is provided with an internal thread;

The cover (10) of the second opening (112) is provided with an internal thread;

a cover (10) of the first opening (111) is screwed onto the first opening (111);

The cover (10) of the second opening (112) is screwed onto the second opening (112).
The islet isolation device according to claim 1, wherein:

The cavity (1) is made of a transparent material.
The islet isolation device according to claim 4, wherein:

The cavity (1) and the cavity connecting cylinder (21) are made of plastic material;

The screen (22) is made of steel.
An islet isolation method using the islet separation device according to any one of claims 1-9, characterized in that:

Includes the following steps:

S1: obtaining a fresh animal pancreas;

S2: Elimination of connective tissue on the pancreas;

S3: configuring a digestive enzyme solution;

S4: placing the pancreas in a petri dish and placing it on an ice bath; using a syringe to remove the enzyme solution, and injecting the digestive enzyme solution into each acinar structure of the pancreas; after the acinus is filled with the digestive enzyme solution, the pancreas is filled Divided into small pieces of tissue;

S5: covering the lid of the first opening (111) on the islet separating device; placing the divided pancreas from the second opening (112) into the first chamber of the islet separating device, and covering the second opening of the islet separating device (112) cover; incubate the islet isolation device in a 37 ° water bath, digest the pancreas; start timing, and gently shake the islet separation device, pancreas digestion for 8-15 minutes; filter mechanism (2) filter through hole diameter 450 μm; opening the lid (10) on the second opening (112), collecting the filtrate; filtering the filtrate through a sieve of 200 μm pore size, collecting the filtrate; centrifuging the filtrate; taking out the cell pellet after centrifugation; The medium was resuspended and the sample was collected for the first time and placed in an ice bath for use.