Human Islet Cell Culture

Islet cell culture is one of the ways to preserve islets in vitro, and can purify islets and reduce immunogenicity. Islet cell culture needs to solve some special problems: firstly, due to the large volume of islet cell clusters, necrosis may occur due to insufficient oxygen supply in the center of the islet; secondly, the evaluation and comparison of islet culture with other cell culture techniques are different; at the same time, islet culture techniques and biological Learning behavior is different. Contamination should be avoided in islet transplantation. Although islet contamination is associated with cryopreserved islets, any additional steps prior to islet transplantation may increase the likelihood of islet contamination.

MCE has not independently verified the accuracy of these methods. They are for reference only.

• Automated Method for Isolation of Human Pancreatic Islets

1. Place the pancreas in Eurocollins solution at 4°C as it was removed from the deceased donor.

2. Cannulate the pancreatic duct with an 18-gauge angiocatheter, inject the Hanks' balanced salt solution containing 2% fetal calf serum and 2 mg/ml collagenase through the pancreatic duct. The amount of collagenase solution injected is approximately equal to double the pancreas weight at 28-30°C.

3. Activate the shaker (320 oscillations/min) and turn on the peristaltic pump with a flow rate of 40 ml/min. Switch on and off the heating circuit bypass to maintain a stable temperature in the digestion chamber, when the temperature in the digestion chamber reach 37°C. Place the cooling circuit and the recirculation cylinder in an ice bath (4°C).

• Islet Cell Purification

1. Load 3-ml aliquots of pelleted tissue from the islet preparation into 250-ml plastic syringes. Pump one hundred milliliters of a Ficoll gradient with a density of 1.074 g/cm³ into each syringe.

2. Suspend the aliquots in 10 ml of Hanks' solution, add to 40 ml of a Ficoll gradient (density = 1.058 g/cm³), and layer over the 1.074-g/cm³ gradient.

3. Centrifuge the syringes 800 x g for 16 min at 4°C. Discard the top layer (20-30 ml) containing cells and fragments.

4. Purified islets exist in the next 50-80 ml of the gradient. The acinar tissue is pelleted in the bottom of the syringe. If the separation is optimal, only a few islets should appear in the acinar pellet.

5. Wash the layer containing purified islets with Hanks'solution by centrifugation at 950 g for 5 min at 4°C. Combine the islet pellets and suspend in 200 ml of tissue culture medium (CMRL-1066) containing 10% fetalcalf serum, penicillin (100 U/ml), streptomycin sulfate (100 μg/ml), D-glucose (1 mg/ml), L-glutamine (2 mM) and HEPES (25 mM). Adjust the medium to pH 7.4 with NaOH.

6. Remove an 0.5-ml aliquot to determine total islet count, islet mass, and the purity of the preparation with a phase microscope fitted with a green filter.

• In Vitro Determination of Islet Function

1. Culture the islets overnight at 37°C in untreated T-flasks. Each flask contains 6000 islets in 50 ml of CMRL-1066.

2. The chambers contains a cellulose acetate Millipore filter. Perifuse the islets at 37°C with Krebs-Ringer bicarbonate solution (0.5% albumin and oxygenated with 95% O₂/5% CO₂)

3. Perifuse the islets with glucose concentrations of 60, 300, and 60 mg/ml for 40 min. The flow rate is 1 ml/min, collect the perifusate with an automatic fraction collector. Freeze the samples for subsequent insulin assay.

• Islet Cell Culture

1. HTB-9 bladder cancer cell line was cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS) and 1 times penicillin-streptomycin.

2. Islet culture medium: Islets were cultured in the medium of CMRL-1066 + 10% fetal bovine serum + 1 times penicillin-streptomycin + 2 mM L-glutamine.

3. Culture HTB-9 cells in T-175 culture flasks to 100% confluency, and continue to culture for 3 days after changing the medium.

4. Collect the conditioned HTB-9 supernatant, filter it with a 0.22 μm membrane filter, and store it at -20°C until use.

5. On the day before the planned inoculation of islet cells, apply 10 μL /well of HTB-9 supernatant to each well of a 384-well plate, and culture overnight at 37°C in 5% carbon dioxide.

6. Wash each well twice with 50 μL /well PBS.

7. Add 10 μL /well islet culture medium and return to the incubator before receiving islets.

8. Upon receipt, centrifuge the pellet in a 50-mL test tube and wash once with PBS.

9. In a 5% carbon dioxide incubator at 37°C, dissociate the islets with Acutase (5000I EQ/mL) for 20min to make the cell colony reach 10 cells. After approximately 10 min, agitate the falcon tube and return to the incubator.

10. Add CMRL diluent (9 ml per 1 ml enzyme dilution).

11. Gently pipette 5-7 times.

12. Suspend the islets in the islet culture medium at a density of 250,000/ml (see above).

13. Islet cells were divided into 10,000 cells/well into coated 384-well culture plates at 37℃ and 5% carbon dioxide.

14. After 14 days of culture on the HTB-9 cell line matrix, there is generally more than 100 times proliferation of islet cells.

1. In the process of cell culture, please pay attention to maintain sterile operation.

2. In the process of passage culture, the digestion time of pancreatic enzyme should not be too long, otherwise it will affect the cell adhesion and growth state.