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WO2019173992A1 - Dispositif et procédé de séparation d'îlots pancréatiques - Google Patents

Dispositif et procédé de séparation d'îlots pancréatiques Download PDF

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Publication number
WO2019173992A1
WO2019173992A1 PCT/CN2018/079062 CN2018079062W WO2019173992A1 WO 2019173992 A1 WO2019173992 A1 WO 2019173992A1 CN 2018079062 W CN2018079062 W CN 2018079062W WO 2019173992 A1 WO2019173992 A1 WO 2019173992A1
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WO
WIPO (PCT)
Prior art keywords
cavity
opening
chamber
islet
pancreas
Prior art date
Application number
PCT/CN2018/079062
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English (en)
Chinese (zh)
Inventor
杜文军
Original Assignee
深圳市云杉医疗管理有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 深圳市云杉医疗管理有限公司 filed Critical 深圳市云杉医疗管理有限公司
Priority to PCT/CN2018/079062 priority Critical patent/WO2019173992A1/fr
Publication of WO2019173992A1 publication Critical patent/WO2019173992A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/12—Apparatus for enzymology or microbiology with sterilisation, filtration or dialysis means

Definitions

  • the invention relates to the field of biomedical technology, and in particular to an islet separating device and method.
  • pancreas 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.
  • pancreatic connective tissue with collagenase, release islet cells, and separate islet cells and exocrine cells by subsequent purification steps
  • 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.
  • the specific separation process requires targeted optimization to establish a suitable method to stably obtain higher quantities and quality of islets.
  • 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.
  • 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.
  • centrifuge tubes 50 ml
  • sieves 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.
  • 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.
  • 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.
  • 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.
  • first opening and the second opening respectively have a cover.
  • the cover is used to seal the cavity.
  • 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.
  • the screen has a disk shape.
  • a plurality of circular filtering through holes are provided on the screen surface of the screen.
  • first spherical cap cavity an internal thread is provided at the junction with the cavity connecting barrel.
  • 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.
  • 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.
  • the cavity is made of a transparent material.
  • the cavity and the cavity connecting barrel are made of a plastic material.
  • the screen is made of steel.
  • volume ratio of the first chamber and the second chamber is between 1:1 and 10:1.
  • the axis of the first opening coincides with the axis of the second opening.
  • An islet isolation method comprising the following steps:
  • 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.
  • 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.
  • Fig. 1 is a schematic perspective view showing the islet separation device of the present invention.
  • FIG. 1 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.
  • 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.
  • 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).
  • 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.
  • the axis of the first opening 111 coincides with the axis of the second opening 112.
  • the volume ratio of the first chamber to the second chamber is between 1:1 and 10:1.
  • 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.
  • 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.
  • 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.
  • opposite sides of the side wall of the cavity connecting cylinder 21 are respectively provided with opposite external threads.
  • an internal thread is provided at the junction with the cavity connecting cylinder 21.
  • 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.
  • 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.
  • 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.
  • 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.
  • the cavity connecting cylinder 21 is made of a plastic material.
  • 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.
  • an islet isolation method which comprises the above-mentioned islet separation device, comprising the following steps:
  • 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);
  • 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;
  • 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.
  • 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 ⁇ m; 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 ⁇ m 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).

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Abstract

Dispositif et procédé de séparation d'îlots pancréatiques. Le dispositif comprend une cavité (1) et un mécanisme filtrant (2). Le mécanisme filtrant divise la cavité interne de la cavité (1) en une première chambre et une seconde chambre, la première chambre étant munie d'une première ouverture (111), la seconde chambre étant munie d'une seconde ouverture (112). Selon le dispositif, la cavité (1) est divisée en deux chambres au moyen du mécanisme filtrant (2). Après un digestat pancréatique dans une chambre, un îlot pancréatique libéré passe de la première chambre à la seconde chambre à travers le mécanisme filtrant (2). La seconde ouverture (112) s'ouvre par intermittence par dévissage pour permettre au fluide pancréatique présente dans la seconde chambre de s'écouler hors de celle-ci, ce qui évite une digestion excessive de l'îlot pancréatique. Le dispositif de l'invention est simple de structure, faible en coût et d'utilisation pratique.
PCT/CN2018/079062 2018-03-14 2018-03-14 Dispositif et procédé de séparation d'îlots pancréatiques WO2019173992A1 (fr)

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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN200943076Y (zh) * 2006-08-07 2007-09-05 王力强 组织消化实验罐及其与温度显示器的组合
US20080009061A1 (en) * 2006-07-05 2008-01-10 Nipro Corporation Method for preserving pancreatic islet, container for preserving pancreatic islet, and kit for transplanting pancreatic islet
CN202482320U (zh) * 2012-01-09 2012-10-10 厚朴生物科技（苏州）有限公司 一种细胞滤器
CN203807470U (zh) * 2014-03-05 2014-09-03 青岛中天干细胞工程有限公司 Svf提取两联袋
CN205741019U (zh) * 2016-03-15 2016-11-30 中国医科大学附属第一医院 一种高效分离纯化胰岛的装置
CN106754251A (zh) * 2017-01-23 2017-05-31 张振 胰岛提取罐及胰岛提取方法
CN108179132A (zh) * 2018-03-14 2018-06-19 深圳海丽施生物医学材料科技有限公司 一种胰岛分离装置及方法

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20080009061A1 (en) * 2006-07-05 2008-01-10 Nipro Corporation Method for preserving pancreatic islet, container for preserving pancreatic islet, and kit for transplanting pancreatic islet
CN200943076Y (zh) * 2006-08-07 2007-09-05 王力强 组织消化实验罐及其与温度显示器的组合
CN202482320U (zh) * 2012-01-09 2012-10-10 厚朴生物科技（苏州）有限公司 一种细胞滤器
CN203807470U (zh) * 2014-03-05 2014-09-03 青岛中天干细胞工程有限公司 Svf提取两联袋
CN205741019U (zh) * 2016-03-15 2016-11-30 中国医科大学附属第一医院 一种高效分离纯化胰岛的装置
CN106754251A (zh) * 2017-01-23 2017-05-31 张振 胰岛提取罐及胰岛提取方法
CN108179132A (zh) * 2018-03-14 2018-06-19 深圳海丽施生物医学材料科技有限公司 一种胰岛分离装置及方法

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