Lentiviral Mediated Gene Silencing in Human Pseudoislet Prepared in Low Attachment Plates

It's difficult to modulate the gene expression in human islets, also greater decline of the functioning human islets pose major challenges after modulation of gene expression. By combining the lentivirus mediated SHR8 transduction and the formation of human suitable islets. This protocol allows efficient downregulation of a targeted gene while preserving islet function during prolonged culture.

To begin, gently swirl the shipping bottle to keep islets in suspension. Transfer the shipping medium containing islets to a 50 milliliter conical centrifuge tube. Let the tube sit in a biological safety cabinet for 15 minutes so that islets settle to the bottom of the tube.

Remove the shipping medium gently using a capillary pipette without disturbing the islet pellet. Re-suspend the islet pellet in CMRL medium with 1%human serum albumin to a concentration of 400 islet equivalent per milliliter. Transfer islets into a non-tissue culture treated dish and culture them at 37 degrees celsius in a 5%carbon dioxide incubator overnight.

After overnight culture, transfer human islets into a 15 milliliter conical centrifuge tube, centrifuge at 250 times g for five minutes in a swinging bucket rotor. And aspirate the medium with a capillary pipette without disturbing the islet pellet. Wash the pellet my adding 10 milliliters of PBS to the tube, mix gently and centrifuge at 250 times g for five minutes.

After centrifugation, aspirate PBS without disturbing the islet pellet. Re-suspend the pellet in 0.5 milliliters of a pre-warmed proteolytic and collagenolytic enzyme mixture. Pipette five times using a P1000 pipette to mix islets.

Incubate at 37 degrees celsius for five minutes. After the incubation, mix pipette petting up and down gently. Check for single cell cloudiness and the number of flakes or undigested islets.

Place a 40 micron strainer in a 35 millimeter petri dish and wet the strainer by adding one milliliter of CMRL medium with 10%heat inactivated fetal bovine serum and depressing with a one milliliter syringe plunger. Transfer all the cell suspension on top of the strainer. And collect the pass through in a fresh 15 milliliter tube.

Wash the tube used for islet digestion with 0.5 milliliters of fresh CMRL medium to collect leftover cells and pass the wash through the strainer. Combine the pass through in a 15 milliliter tube. Repeat once.

To dissociate undigested islets remaining on the strainer, press the strainer with a one milliliter syringe plunger. Collect the pass through again and wash the strainer with fresh plain CMRL 1066 medium to remove all remaining digested islets from the strainer and the dish. Add the pass through to the 15 milliliter tube.

Record the total volume of the cell suspension and take a 10 microliter aliquot of cells to count the cell number on a hemocytometer. After centrifuging the cell suspension for five minutes at 200 times g, remove the medium without disturbing the pellet. To produce pseudo-islets using a 96 well ultra low attachment plate, first calculate the total volume of the required one times 10 to the fifth cells per milliliter of the single cell suspension.

Then, re-suspend the islet pellet in CMRL medium with 10%heat inactivated fetal bovine serum so that 30 microliters of the cell suspension has 3000 cells. Next, transfer the required volume of the single cell suspension to a fresh 15 milliliter tube. At 250 transduction units per cell of a lentivirus containing SH-RNA targeting a gene of interest or a control.

To mix the cell suspension with the virus, pipette gently five times with a P1000 pipette. Then transfer the mixed cells into a 50 milliliter sterile reagent reservoir if using an eight channel pipette. With an eight channel pipette or a P200 pipette, dispense 30 microliters per well of the cell suspension mixed with lentivirus into each well.

Next, centrifuge the 96 well plate in a swinging bucket plate centrifuge at 270 times g at room temperature for seven minutes. Culture at 37 degrees celsius in a humidified 5%carbon dioxide incubator overnight. After incubation, remove the 96 well plate from the incubator and place it in the biological safety cabinet.

Pipette 100 microliters per islet of 10%high FBSCMRL into a well to prevent drying of islets and continue to culture for one week. Pipette 100 microliters per well of CMRL medium with 10%heat inactivated fetal bovine serum from the reservoir to the pseudo-islets and pipette up and down two to three times gently in the well to lift islets up. Then, aspirate the medium in the well containing a pseudo-islet and eject into a 10 centimeter petri dish.

Check the plate under a light microscope to ensure the complete removal of all the pseudo-islets and proceed to downstream experiments. To produce pseudo-islets using a 24 well micro well culture plate, first add 500 microliters per well of an anti-adherence rinsing solution. Centrifuge at 1300 times g for five minutes in a swinging bucket plate centrifuge.

Then, observe the plate under a microscope to ensure that air bubbles are removed from micro-wells. In a biological safety cabinet, aspirate the anti-adherence rinsing solution from the wells. Rinse each well with two milliliters of warm plain CMRL 1066 medium once.

Then, aspirate the plain CMRL 1066 medium and add 0.5 milliliter per well of warm CMRL with 10%heat inactivated fetal bovine serum to each well planned for use. Determine the total number of cells needed for each well. Re-suspend the single cells in 0.8 milliliters of CMRL medium with 10%heat inactivated fetal bovine serum in a sterile 1.5 milliliter tube.

To create one well of pseudo-islet transduced by a lentivirus, add 125 transduction units per cell to the single cell suspension keeping the volume of virus below 0.2 milliliters. Incubate the cell and virus mixture at 37 degrees celsius with occasional gentle mixing for one hour to allow contact of cells with virus before condensation of cells. After one hour, adjust the total volume of the islet cell and virus mixture to one milliliter by adding CMRL medium with 10%heat inactivated fetal bovine serum.

If cells form clumps after a one hour incubation disperse into a single cell suspension by gentle and quick pipetting two to three times. Transfer the cell suspension to one well of the 24 well micro-well culture plate. Immediately following pipetting, centrifuge at 100 times g at room temperature for three minutes to capture cells into all micro-wells.

Observe under a microscope to verify that cells are evenly distributed in all micro-wells. It's critical to centrifuge the micro-well plate immediately after adding the well mixing the islet single cell suspension into the wells. Sequential changes in morphology of pseudo-islets from 3000 human islet cells created in a 96 well ultra low attachment plate showed monolayer or loose clumps of cells turned into solid aggregates with a smooth round border in one week.

In contrast, in a micro-well culture plate, the formation of cell and pseudo-islets from only 500 islet cells is usually visible within four days. The uniform size of pseudo-islets reduces the variation within a test group and allows static incubation using as little as five pseudo-islets per measurement. The Perry Fusion Assay for insulin response showed human pseudo-islets maintained robust first phase insulin secretion in response to glucose after seven days of culture.

The introduction of lentivirus targeting human adipose triglyceride lipase or perilipin five genes into a single cell suspension ensures the efficient and homogenous transduction of islet cells and achieves highly efficient downregulation of genes. It is important to remember that the time required for dispersing human islet into single cells is affected by many factors such as:size, distribution, and the donor characteristic. So, it is important to pay close attention to the disappearance of human islets during digestion.

Throughout the protocol it is important to handle single cell suspension gently to avoid cell loss. The human pseudo-islets being created can be used for many applications to determine the effect of gene modulation such as:Western Blot, histological study and the measurements of oxygen consumption rates. This protocol allows assess the low of targeted gene in the regulation of islet health and the function in human islets by using common lab ware and simple techniques.

The use of human islets may require a prior approval from a local review board. Please consult local review board before initiation of the study. Lentivirus is classified as biosafety level two.

Contact biosafety committee before the initiation of the use of the lentivirus.