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WO2018105805A1 - Procédé d'isolement d'un îlot de porc nouveau-né - Google Patents

Procédé d'isolement d'un îlot de porc nouveau-né Download PDF

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Publication number
WO2018105805A1
WO2018105805A1 PCT/KR2017/000273 KR2017000273W WO2018105805A1 WO 2018105805 A1 WO2018105805 A1 WO 2018105805A1 KR 2017000273 W KR2017000273 W KR 2017000273W WO 2018105805 A1 WO2018105805 A1 WO 2018105805A1
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Prior art keywords
islets
islet
npccs
newborn
enzyme
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PCT/KR2017/000273
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English (en)
Korean (ko)
Inventor
안규리
이은미
자히드알람
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SNU R&DB Foundation
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Seoul National University R&DB Foundation
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0676—Pancreatic cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/02—Atmosphere, e.g. low oxygen conditions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/60—Buffer, e.g. pH regulation, osmotic pressure
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/70—Enzymes
    • C12N2501/73—Hydrolases (EC 3.)
    • C12N2501/734—Proteases (EC 3.4.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2523/00—Culture process characterised by temperature

Definitions

  • the present invention relates to a method for separating islets from newborn pigs, specifically (a) extracting the islets from newborn pigs; (b) injecting enzyme into the isolated pancreas; And (c) relates to a method for isolating islets of newborn pigs comprising the step of inducing the activation of the enzyme.
  • pancreatic islet transplantation is a relatively simple treatment, less surgical complications, and can be transplanted repeatedly, which is particularly applicable to patients with type 1 diabetes.
  • pancreatic islet transplantation is a relatively simple treatment, less surgical complications, and can be transplanted repeatedly, which is particularly applicable to patients with type 1 diabetes.
  • pancreatic islets due to their rapid development and maturity, large numbers of offspring, organ size and physiological functions similar to humans, and the secretion of biologically active insulin in humans (Dufrane). et al., Transplant Rev (Orlando), 26 (3): 183-8, 2012).
  • the study of the clinical application of xenograft islets to humans transplanting pig islets is considered an alternative to diabetes treatment (Samy et al., Xenotransplantation, 21 (3): 221-9, 2014).
  • a technique for isolating high quality pig islets is an essential technique for xenograft transplantation, and research on this is required.
  • the present inventors have diligently tried to develop an optimal pancreatic isolating technique, and as a result, it has been developed that the separation and culture and degree analysis technology of the new pig islets can be used to isolate high quality pancreatic islets with high yield.
  • the present invention has been completed.
  • the object of the present invention is to (a) extract the islets from the newborn pig; (b) injecting enzyme into the isolated pancreas; And (c) to provide a method for isolating islets of newborn pigs comprising the step of inducing the activation of the enzyme.
  • the present invention comprises the steps of (a) extracting the islets from the newborn pig; (b) injecting enzyme into the isolated pancreas; And (c) provides a method for isolating islets of newborn pigs comprising the step of inducing the activation of the enzyme.
  • pancreatic islet separation technique for cross-transplantation, as well as isolation and culture and degree of newborn pig islets.
  • analytical techniques they can be widely used in biomedical fields such as diabetes treatment.
  • Figure 1 shows the representative results observed after culturing the isolated NPCCs form by microscopic visual inspection for 1 day, 4 days and 7 days.
  • Figure 2 shows the results of ATP activity analysis to confirm the viability of NPCCs.
  • FIG. 3 shows the results of glucose-stimulated insulin secretion (GSIS) analysis to measure the insulin secretion function of NPCCs.
  • GSIS glucose-stimulated insulin secretion
  • the present invention relates to a method for separating high quality pancreatic islets from newborn pigs in high yield.
  • the present invention in one aspect, (a) extracting the islets from the newborn pig; (b) injecting enzyme into the isolated pancreas; And (c) relates to a method for isolating islets of newborn pigs comprising the step of inducing the activation of the enzyme.
  • the enzyme may be characterized in that the collagenase V (Sigma-Aldrich, MO, USA).
  • the step (c) is preferably shaken in a 37 °C water bath may be characterized in that the activation of the enzyme.
  • step (c) may be characterized in that it further comprises the step of culturing the islets, preferably characterized in that incubated in the presence condition of 5% carbon dioxide at 37 °C for 1 to 14 days. can do.
  • F10 medium It may also be characterized by culturing in F10 medium.
  • the composition of the F10 medium is shown in Table 1 below.
  • the Hank balanced salt solution may be used as a buffer.
  • the composition of the HBSS is shown in Table 2 below.
  • Substance Tinnitus (common name) CAS number content(%) Calcium chlorate 10035-04-8 0.01 Magnesium Sulfate Nitrate Magnesium Sulphate, Heptahydrate 10034-99-8 0.001 Potassium chloride Potassium chloride 7447-40-7 0.004 Potassium phosphate monobasic Potassium Acid Phosphate 7778-77-0 0.006 Sodium chloride Sodium chloride 7647-14-5 0.8 Sodium Phosphate, Dibasic Disodium phosphate 7558-79-4 0.005 Dextrose D-glucose 50-99-7 0.1 Sodium bicarbonate Sodium bicarbonate 144-55-8 0.04 Penicillin-streptomycin, liquid 3810-74-0; 69-57-8 0.5 albumin Albuminz, Blood Serum 9048-46-8 0.25 Phenolic Red, Sodium Salt 34487-61-1 0.001 HEPES, free acid N- (2-hydroxyethyl) piperazine-N '-(2-
  • the newborn pig may be characterized as being three days to five days old, but is not limited thereto.
  • NPCCs Neonatal Porcine Pancreatic Cell Clusters
  • Pancreatic cell clusters were isolated from 3 to 5 day old piglets (Landrace or Yorkshire), weighing approximately 1.0 to 2.0 kg. Several modifications have been made to known separation methods to isolate NPCCs (Korbutt GS et al., The Journal of clinical investigation, 97 (9): 2119-29, 1996).
  • Newborn pigs were sequentially administered 0.1 mg / kg of azaferon (Stresnil, Janssen, Geneva, Belgium), 125 mg / kg of tiletamine hydrochloride, and zolazepam hydrochloride (Zoletil, Virbac, Carros, France). .
  • a midline incision was used to open the newborn pigs for pancreatic exposure.
  • the pancreas was carefully dissected from the surrounding pylorus, duodenum and artery. Special care has been taken to prevent bacterial infection, which may be caused by bowel nipping.
  • the pancreas was cut into small pieces (1-2 mm 3 ) and Hank's balanced salt solution (Hank's) supplemented with 0.5% penicillin and streptomycin (Invitrogen, Carlsbad, USA), 0.25% bovine serum albumin (BSA) and 12.5 mM HEPES. rinsed in a balanced salt solution (HBSS).
  • the tissues were then digested for exactly 11 minutes in a 37 ° C. water bath using 1.0 mg / mL collagenase V (Sigma-Aldrich, MO, USA). All the digested tissues were shaken vigorously by hand for 30 seconds until the color became milky white, and then the HBSS was added to stop the enzyme activity.
  • the digested tissue was centrifuged at 450 xg for 1 minute. After resuspension, the degraded tissue was filtered through a stainless steel mesh (500 ⁇ m). The filtrate containing NPCCs was rinsed twice in HBSS. Finally, islet suspensions were supplemented with 10% adult porcine serum (APS; Gibco, Cat.No. 26250-084), 2% BSA, 0.1% ciprofloxacin and 1% penicillin and streptomycin (Ham's) F10 medium 37 Incubated in humidified air containing 5% CO 2 in the incubator. The islet islet suspension was placed in a 150 mm Petri dish for 7 days prior to the experiment. Culture medium was changed every other day. An algorithm was used to calculate 150 ⁇ m diameter pancreatic equivalent number (IEQ) (Cardona K et al., Nature medicine, 12 (3): 304-6, 2006).
  • IEQ 150 ⁇ m diameter pancreatic equivalent number
  • Isolated NPCCs were observed after culturing for 1, 4 and 7 days after separation using a microscope (Leica DFC 295) (FIG. 1). The average weight of the isolated islets was 1.73 ⁇ 0.45g.
  • NPCCs 100 IEQ / well in 100 ⁇ l volume
  • NPCCs 100 IEQ / well in 100 ⁇ l volume
  • NPCCs 100 IEQ / well in 100 ⁇ l volume
  • NPCCs 100 IEQ / well in 100 ⁇ l volume
  • NPCCs 100 IEQ / well in 100 ⁇ l volume
  • NPCCs 100 IEQ / well in 100 ⁇ l volume
  • NPCCs 100 IEQ / well in 100 ⁇ l volume
  • Cell Titer-Glo reagent 319
  • Cells were incubated for 30 minutes at room temperature to stabilize the luminescence signal.
  • Luminescence was then recorded with a luminescence counter (VICTOR TM Light, PerkinElmer, Mass., USA).
  • Luminescence indicates the metabolic activity of the cell. More than 90% (18 times / 20 times) of the separated NPCCs showed more than the reference value (red dotted line in FIG. 2) of the emission value corresponding to the NPCCs 100 IEQ,
  • Example 3 Of NPCCs For measuring insulin secretory response rate Glucose -Stimulated insulin secretion ( GSIS ) analysis
  • NPCCs insulin secretion response rate of the isolated NPCCs
  • cells were cultured for 2 weeks and then subjected to GSIS analysis.
  • the isolated islets were incubated on glucose at various concentrations.
  • NPCCs were suspended and pre-incubated for 30 minutes in Krebs Ringer buffered HEPES (KRBB, pH 7.4).
  • NPCCs were continuously incubated in low glucose (2.5 mM) buffer and then high glucose (20 mM) buffer in humidified air containing 5% CO 2 in 37 incubators for 1 hour.
  • low glucose solution 2.5 mM
  • high glucose solution 20 mM
  • the concentration of insulin protein secreted in the buffer was measured using the DIAsource INS-Irma kit (DIAsource ImmunoAssays SA, Louvain-la-Neuve, Belgium) (Schroff RW et al., Cancer research, 45 (2): 879-85, 1985).
  • the stimulation index (SI) value was calculated by dividing the amount of insulin secreted in the high glucose solution by the amount in the low glucose solution (Jung HS, Official journal of the Controlled Release Society, 172 (3): 1092-101, 2013). As a result, it was confirmed that the range of SI values was 1 to 1.5, which indicates normal insulin secretory function in response to different glucose stimuli (FIG. 3).
  • NPCCs were separated by approximately 13,000 IEQ (13,539.45 ⁇ 5182.54 IEQ) per gram of pancreatic tissue (Table 3).
  • NPCCs The parameters for the separation of NPCCs are as follows: date of separation, number of newborn pigs, age of newborn pig, weight of newborn pig, weight of pancreas (time / new pig), pancreatic weight before digestion, collagenase type (Cat.No. or Lot). No.), collagenase concentration, degradation time, total cell count measured on day 7, NPCCs yield (IEQ / new pig or IEQ / pancreatic weight gram) (Table 4).
  • HTK solution containing 1% penicillin-streptomycin (P / S; 10,000 U / mL) and 1% gentamycin sulfate: lL of HTK solution was obtained from the infusion packet. After ingestion, 10 ml of penicillin and streptomycin (P / S) are added, 10 ml of gentamycin sulfate is added, and the samples are subdivided into tubes every 20 ml and stored in an ice box for the extracted pancreas.
  • betadine in saline To 700 ml of saline solution, 300 ml of betadine (Povidone-iodine, Mundi Pharma) is added and then subdivided into 20 ml tubes to wash the extracted pancreas.
  • Penicillin and streptomycin (Gibco, Cat. No. 15140-122).
  • Ketamine (Imalgenem Gellini Farma-ceutici, Aprilia, Italy).
  • PAD (Cat. No. B414A003-7011).
  • the leaf paper The leaf paper.
  • the isolated pancreas is stored in HTK solution and placed in an ice box until the separation of NPCCs begins.
  • F-10 medium (pH 7.3) containing 10% adult pig serum, 1% P / S and 0.1% ciprofloxacin (Biosesang, Cat. No. F2014).
  • Collagenase type V (collagenase derived from Clostridium histories) (Sigma, Cat. No. C9263).
  • Steriplip-GP Filter Unit (0.22 ⁇ m) (MILLIPORE, Cat.No. SCGP00525).
  • the extracted pancreas is placed in an empty conical tube and the individual pancreas is weighed (keep the same mark).
  • the suspension is then passed through a 500 ⁇ m mesh sieve, rinsing the walls of each tube and again passing 30 ml HBSS through the mesh sieve.
  • NPCCs isolated from one conical tube are divided into four 150 mm Petri dishes (medium volume 35 ml).
  • NPCCs are obtained from four Petri-plates with four conical tubes.
  • NPCCs are obtained from four Petri-plates with four conical tubes.
  • pancreatic islet separation technique for cross-transplantation, as well as isolation and culture and degree of newborn pig islets.
  • analytical techniques they can be widely used in biomedical fields such as diabetes treatment.

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Abstract

La présente invention concerne un procédé d'isolement d'un îlot de porc nouveau-né et, plus spécifiquement, un procédé d'isolement d'un îlot de porc nouveau-né, le procédé comprenant les étapes suivantes : (a) extraire un îlot d'un porc nouveau-né ; (b) injecter une enzyme dans le pancréas ayant fait l'objet de l'extraction ; et (c) induire l'activation de l'enzyme. L'utilisation du procédé d'isolement d'un îlot selon la présente invention permet d'isoler un îlot de grande qualité d'un porc nouveau-né à un rendement élevé, et peut ainsi conduire à non seulement son utilisation à titre de technique d'isolement d'îlots pour xénogreffe, mais aussi au développement de techniques d'isolement, de culture et d'analyse de qualité d'un îlot de porc nouveau-né, et par conséquent, le procédé selon la présente invention peut être largement utilisé dans le domaine biomédical, tel que le traitement du diabète.
PCT/KR2017/000273 2016-12-08 2017-01-09 Procédé d'isolement d'un îlot de porc nouveau-né Ceased WO2018105805A1 (fr)

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KR10-2016-0166487 2016-12-08
KR1020160166487A KR101914837B1 (ko) 2016-12-08 2016-12-08 신생돼지의 췌도 분리 방법

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114081694A (zh) * 2021-12-15 2022-02-25 赛诺医疗科学技术股份有限公司 血管降解溶液、动物试验后血管内支架的获取方法及应用

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997039107A2 (fr) * 1996-04-12 1997-10-23 The Governors Of The University Of Alberta Procede permettant d'accelerer la maturation des cellules
KR100686383B1 (ko) * 2005-08-17 2007-02-22 피더블유제네틱스코리아 주식회사 이종이식을 위한 돼지의 췌도세포 분리방법
KR20070095956A (ko) * 2004-12-22 2007-10-01 고쿠리츠 다이가쿠 호진 교토 다이가쿠 췌장 소도의 분리방법
KR20130091260A (ko) * 2010-07-13 2013-08-16 가부시키가이샤 오츠카 세이야쿠 고죠 췌도의 분리방법 및 췌도조직을 보호하기 위한 보호액

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997039107A2 (fr) * 1996-04-12 1997-10-23 The Governors Of The University Of Alberta Procede permettant d'accelerer la maturation des cellules
KR20070095956A (ko) * 2004-12-22 2007-10-01 고쿠리츠 다이가쿠 호진 교토 다이가쿠 췌장 소도의 분리방법
KR100686383B1 (ko) * 2005-08-17 2007-02-22 피더블유제네틱스코리아 주식회사 이종이식을 위한 돼지의 췌도세포 분리방법
KR20130091260A (ko) * 2010-07-13 2013-08-16 가부시키가이샤 오츠카 세이야쿠 고죠 췌도의 분리방법 및 췌도조직을 보호하기 위한 보호액

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
PARK, SOL JI ET AL.: "Functional Improvement of Porcine Neonatal Pancreatic Cell Clusters via Conformal Encapsulation Using an Air-driven Encapsulator", EXPERIMENTAL AND MOLECULAR MEDICINE, vol. 44, no. 1, January 2012 (2012-01-01), pages 20 - 25, XP055510111 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114081694A (zh) * 2021-12-15 2022-02-25 赛诺医疗科学技术股份有限公司 血管降解溶液、动物试验后血管内支架的获取方法及应用

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