WO2018105805A1 - Method for isolating islet of newborn pig - Google Patents

Abstract

The present invention relates to a method for isolating an islet from a newborn pig and, specifically, to a method for isolating an islet of a newborn pig, the method comprising the steps of: (a) extracting an islet from a newborn pig; (b) injecting an enzyme into an extracted pancreas; and (c) inducing the activation of the enzyme. The use of the method for isolating an islet according to the present invention can isolate a high-quality islet from a newborn pig at high efficiency, and thus can lead to not only the utilization as an islet isolation technique for xenotransplantation but also the development of techniques for isolation, culture, and quality analysis of an islet of a newborn pig, and therefore, the method according to the present invention can be widely utilized in the biomedical field, such as diabetes treatment.

Description

How to isolate islets of newborn pigs

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.

Diabetes is a leading global disease that affects about 422 million adults in 2014. Since 1980, the prevalence has nearly doubled, rising from 4.7% to 8.5% of the adult population, according to the 2016 WHO Global Diabetes Report 2016. Among various therapies for insulin-dependent diabetes, pancreatic islet transplantation has been widely recognized as one of the promising therapies for insulin-dependent diabetes patients. 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. However, due to the large shortage of long-term donors, long-term supply and demand are not balanced.

On the other hand, pigs are considered an ideal source of 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). Thus, 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).

In 1997, Dinsmore et al. Identified whether there is a difference in the separation efficiency of pancreatic islets and the function of expressing insulin according to the types of enzymes used in the isolation process to establish a technique for isolating fetal islets. (US 5629194B2), Korbutt et al. Reported that fetal pig islet cell aggregates were isolated and cultured in vitro for 9 days before transplanting under the neocapillary membrane of diabetic mice to regulate blood glucose control. (Korbutt GS et al., The Journal of clinical investigation, 97 (9): 2119-29, 1996), and Lamb et al. Used an average 20-day-old pig to isolate the pancreatic islets and perform a degree analysis. (M. Lamb et al., Cell transplantation, 23 (3): 263-72, 2014).

As such, a technique for isolating high quality pig islets is an essential technique for xenograft transplantation, and research on this is required.

Therefore, 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.

In order to achieve the above object, 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.

Using the method for isolating islets according to the present invention can separate high-quality pancreatic islets from newborn pigs with high efficiency, and can be used as a pancreatic islet separation technique for cross-transplantation, as well as isolation and culture and degree of newborn pig islets. By developing 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.

Figure 3 shows the results of glucose-stimulated insulin secretion (GSIS) analysis to measure the insulin secretion function of NPCCs.

4 is a NPCCs number calculation chart.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.

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.

In the present invention, the enzyme may be characterized in that the collagenase V (Sigma-Aldrich, MO, USA).

In the present invention, the step (c) is preferably shaken in a 37 ℃ water bath may be characterized in that the activation of the enzyme.

In the present invention, after the 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 ℃ for 1 to 14 days. can do.

It may also be characterized by culturing in F10 medium. The composition of the F10 medium is shown in Table 1 below.

Substance	Tinnitus (common name)	CAS number	content(%)
Nutrient Mixture F-10 Ham	Ham's F-10		87
Sodium bicarbonate	Sodium bicarbonate	144-55-8	0.1
Nicotinamide	Niacinamide: Vitamin B3	98-92-0	0.1
Dextrose	D-glucose	50-99-7	0.07
L-(+)-Glutamine	2-aminoglutamic acid	56-85-9	0.01
Calcium chlorate, dihydrate	Calcium chlorate (II), calcium chlorate, E509	10035-04-8	0.02
albumin	Albuminz, Blood Serum	9048-46-8	2
3-isobutyl-1-methylxanthine	3-isobutyl-1-methyl-2,6 (1H, 3H) -purindione	28822-58-4	0.001
Penicillin-streptomycin, liquid		3810-74-0; 69-57-8	One
Ciprofloxacin	Ciproby	85721-33-1	0.002
Pig serum			10

In the present invention, 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-ethanesulfonic acid)	7365-45-9

In the present invention, the newborn pig may be characterized as being three days to five days old, but is not limited thereto.

EXAMPLE

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Example 1: Isolation and Identification of Neonatal Porcine Pancreatic Cell Clusters (NPCCs)

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, Bruxelles, 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 μm). 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 μm diameter pancreatic equivalent number (IEQ) (Cardona K et al., Nature medicine, 12 (3): 304-6, 2006).

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.

The protocol for animal use was approved by the Seoul National University Laboratory Animal Steering Committee (SNU-151103-1) in accordance with Seoul National University's Guidelines for the Protection and Use of Laboratory Animals.

Example 2: ATP Activity Assay for Confirmation of NPCCs Viability

Measurement of intracellular ATP has been demonstrated as a simple and reliable indicator of cell viability, including metabolic function of islet cells (Ishii S et al., Transplantation proceedings, 37 (8): 3499-500, 2005). Cell viability assays were performed by quantitative assessment of ATP in 7-day cultures.

Cell viability was quantitated using the Luminescent Cell Viability Assay Kit (Promega Corporation, Madison, USA). NPCCs (100 IEQ / well in 100 μl volume) were placed in 96-well plates and the plates and their contents were taken out beforehand to room temperature. To induce cell lysis, equal volumes of Cell Titer-Glo reagent (39) were added and mixed for 2 minutes. Cells were incubated for 30 minutes at room temperature to stabilize the luminescence signal. Luminescence was then recorded with a luminescence counter (VICTOR ^™ 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, thereby confirming the viability of normal metabolism (FIG. 2). .

Example  3: Of NPCCs  For measuring insulin secretory response rate Glucose -Stimulated insulin secretion ( GSIS ) analysis

In order to measure the 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. When isolated NPCCs were continuously incubated for 1 hour in a low glucose solution (2.5 mM) or high glucose solution (20 mM), the insulin secretion pattern was very similar. Finally, 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).

Example 4 Confirmation of NPCCs Separation Yield

After incubating the isolated NPCCs for 7 days, the separation yield of NPCCs was confirmed. It was confirmed that NPCCs could be separated by approximately 13,000 IEQ (13,539.45 ± 5182.54 IEQ) per gram of pancreatic tissue (Table 3).

TABLE 3

Example  5: newborn pig Pancreatic islet  Establishment of Standard Operating Procedures (SOPs) for Separation, Culture and Quality Analysis Methods

5-1: NPCCs  Parameters for Separation

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).

TABLE 4

5-2: Pancreas Extraction

5-2-1: Material

HTK solution containing 1% penicillin-streptomycin (P / S; 10,000 U / mL) and 1% gentamycin sulfate: lL of HTK solution (Dr. Frank Kohler Chemie GmbH, Cat. No. D64625) 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.

30% 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.

Subdivide the normal salt solution (20ml / tube) to wash the extracted pancreas.

Penicillin and streptomycin (Gibco, Cat. No. 15140-122).

Gentamycin sulfate (Biowest, Cat. No. L0011-100).

K-40 (JW Pharmacuticals, Cat. No. 13011).

Scrub (veratin scrub, Dongin dang Pharmaceutical Co., Ltd.).

Ketamine (Imalgenem Gellini Farma-ceutici, Aprilia, Italy).

Stressil (Janssen Pharmaceuticals, Cat.No. CAB0800).

PAD (Cat. No. B414A003-7011).

Surgical gown (DOWOO, KM Healthcare Corporation Ltd., GWN-102).

Half sheet (DOWOO, KM Healthcare Corporation Ltd., WRD-1118).

Surgical equipment set

Steril gauze (Dae Han medical Supply Co. Ltd., Cat.No. GST315002).

Surgical Gloves (Cardincal Health, Cat.No. 2D7203I).

General Hand Gloves (Complete Care Plus).

50 ml conical tubes (Greenpia Techinology, Cat. No. 50050).

Syringes (1 ml, 3 ml, and 30 ml) from Korea Vaccine Co. Ltd.

Surgical knife (Feather Disposable Sterile Surgical Blades, No. 20).

Needle (17G) (Korea vaccine Co. Ltd.)

70% ethanol (Duksan, Cat. No. 64-17-5).

The leaf paper.

5-2-2: Course

1. First, repair the surgery area.

2. Inject anesthetic into the muscle (lower arm) before sacrifice (0.1 mg / kg of stressil (azaferon) and 15 mg / kg of ketamine).

3. Measure and record your weight.

4. Wash the new pig body with tap water, sprinkle 70% ethanol and wipe it with thin paper.

5. Puncture the heart to obtain blood.

6. Inject K-40 into the heart via the same route (approximately 3 ml).

7. Clean the entire body with a scrub.

8. Incision in newborn pig abdomen.

9. Extract the pancreas (using steryl gauze and fingers). The pancreas is then weighed.

10. Lightly soak in 30% betadine solution and immediately wash with brine.

11. The isolated pancreas is stored in HTK solution and placed in an ice box until the separation of NPCCs begins.

5-3: NPCCs Separation

5-3-1: Material

Hank's balanced salt solution (pH 7.35) containing 0.25% BSA, 0.5% P / S (Biosesang, Cat. No. H2074).

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).

Forceps and scissors (autoclave).

500 μm mesh sieve.

Steriplip-GP Filter Unit (0.22μm) (MILLIPORE, Cat.No. SCGP00525).

50 ml conical tubes.

Alcohol lamp.

10 ml and 25 ml pipettes and pipette-aids.

5-3-2: Course

1. Soak F-10 medium and HBSS in 37 ℃ constant temperature water bath.

2. Prepare the pancreas extracted in step 5-5-2.

3. Set up a sterile bench to isolate NPCCs (subsection: turn on HBSS, 100% ethanol, alcohol lamp).

4. Place the sterilized tweezers into a small HBSS tube.

5. The extracted pancreas is placed in an empty conical tube and the individual pancreas is weighed (keep the same mark).

6. Add 15 ml of HBSS to each tube (per 1 gram pancreas).

7. Scissor continuously in a tube for 30 seconds to form a small mass. Adjust the volume to 50 ml by adding HBSS. Slightly use the scissors in 100% ethanol, dry in an alcohol lamp, lightly soak in HBSS and then again. As used, disinfect the scissors every time.

8. Centrifuge (1500 rpm, 1 min).

9. Discard the supernatant up to the cell pellet.

10. Prepare collagenase solution (1 mg / ml), filter with a 0.22 μm filter and mix 25 ml of the prepared collagenase solution to the cut pancreas (25 ml collagenase solution / 1 gram equivalent of pancreas).

11. Seal the lid of the conical tube containing each enzyme and pancreas and incubate for 11 minutes in a 37 ° C shaker bath (shaking speed 190 rpm).

12. Then shake your hands strongly for 30 seconds.

13. Adjust volume to 45 ml with cold HBSS to stop enzymatic activity.

14. Centrifuge (1500 rpm, 1 min).

15. Discard the supernatant up to the cell pellet and gently resuspend with 20 ml HBSS.

16. The suspension is then passed through a 500 μm mesh sieve, rinsing the walls of each tube and again passing 30 ml HBSS through the mesh sieve.

17. Centrifuge (1500 rpm, 1 min).

18. Discard the supernatant up to the cell pellet, gently resuspend with 20 ml HBSS and finally adjust to 45 ml.

19. Turn the tube upside down 3 times and centrifuge (1500 rpm, 1 min).

20. Discard the supernatant up to the cell pellet.

21. Add F-10 medium (35 μl exenatide-4 mixed in 35 ml medium, concentration 20 μM) supplemented in cell pellet and resuspend appropriately.

22. NPCCs isolated from one conical tube are divided into four 150 mm Petri dishes (medium volume 35 ml).

23. Incubate in a 37 ° C. cell culture in order to continue the next culture.

5-4: NPCCs culture (1-7 days)

5-4-1: Process

1. Soak F-10 medium and HBSS in 37 ℃ constant temperature water bath.

2. Set up a sterile bench and subtract HBSS to wash NPCCs.

3. Take NPCCs culture dish from cell incubator and check cell morphology under microscope.

4. Obtain all NPCCs contained in the medium into tubes in a petri-dish (first obtained and transferred to conical tubes, secondly rinsed petri-dish twice with HBSS (7ml / each); NPCCs are obtained from four Petri-plates with four conical tubes.

5. Centrifuge (1500 rpm, 1 min, braking step 4).

6. Discard the supernatant until it is about 5 ml in volume.

7. Resuspend each tube and collect in one tube (collect all NPCC pellets in one tube).

8. Adjust to 45 ml volume with HBSS and centrifuge (1500 rpm, 1 min, braking step 4).

9. Discard the supernatant to pellet volume and properly resuspend with supplemented medium (Exenatide-4), then divide into four 150 mm Petri-plates (medium volume 35 ml / dish).

10. Incubate in a 37 ° C. cell culture in order to continue the next culture.

※ Follow the same NPCCs culture as above: 3 days → 5 days → 7 days (total cell count).

5-5: Count NPCCs (day 7)

5-5-1: Cell Preparation

1. Soak F-10 medium and HBSS in 37 ℃ constant temperature water bath.

2. Set up a sterile bench and subtract HBSS to wash NPCCs.

3. Take NPCCs culture dish from cell incubator and check cell morphology under microscope.

4. Obtain all NPCCs contained in the medium into tubes in a petri-dish (first obtained and transferred to conical tubes, secondly rinsed petri-dish twice with HBSS (7ml / each); NPCCs are obtained from four Petri-plates with four conical tubes.

5. Centrifuge (1500 rpm, 1 min, braking step 4).

6. Discard the supernatant until it is about 5 ml in volume.

7. Resuspend each tube and collect in one tube (collect all NPCC pellets in one tube).

8. Adjust to 45 ml volume with HBSS and centrifuge (1500 rpm, 1 min, braking step 4).

9. Discard supernatant up to 5 ml volume, gently resuspend and adjust to 40 ml volume for cell counting.

5-5-2: Course

1. Pour 4 ml HBSS into a new 50 ml conical tube.

2. Obtain 1 ml cell suspension from the final suspension and add to 4 ml HBSS (5 ml final dilution volume).

3. Gently shake with your left hand to obtain 100 μl cell suspension and line 60 mm Petri-dish for cell counting.

4. Calculate the number (IEQ) according to the NPCCs number calculation chart (Figure 4).

The specific parts of the present invention have been described in detail above, and it is apparent to those skilled in the art that such specific descriptions are merely preferred embodiments, and thus the scope of the present invention is not limited thereto. something to do. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Using the method for isolating islets according to the present invention can separate high-quality pancreatic islets from newborn pigs with high efficiency, and can be used as a pancreatic islet separation technique for cross-transplantation, as well as isolation and culture and degree of newborn pig islets. By developing analytical techniques, they can be widely used in biomedical fields such as diabetes treatment.

Claims (8)

1. How to isolate islets of newborn pigs comprising the following steps:

   (a) extracting the islets from the newborn pigs;

   (b) injecting enzyme into the isolated pancreas; And

   (c) inducing activation of the enzyme.
2. The method of claim 1, wherein the enzyme is collagenase V, islet separation method of neonatal pigs.
3. According to claim 1, wherein the step (c) is shaken in a 37 ℃ water tank, characterized in that to induce the activation of the enzyme, islet separation method of newborn pigs.
4. The method of claim 1, further comprising culturing the islets after the step (c).
5. The method for separating islets of neonatal pigs according to claim 4, wherein the piglets are cultured at 37 ° C under 5% carbon dioxide.
6. The method for separating islets of neonatal pigs according to claim 4, which is cultured in F10 medium.
7. The method of claim 1, wherein Hank's balanced salt solution (HBSS) is used as a buffer.
8. The method of claim 1, wherein the newborn pigs are 3 to 5 days old.

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