Hello, I'm Florian Al from the Department of Surgery at the Technical University in Triton. Hi, I'm Gregor Cher, also from the Department of Surgery at the University Hospital. In Grayston.
Research on eyelid biology and function is desperately needed to advance our understanding of eyelid physiology and diabetes pathogenesis. Unfortunately, human eyelids from non-diabetic and type two diabetic patients are very difficult to obtain. The following Video sequence will show you a protocol how to isolate human eyelids from partial pancreatic optimiz patients.
The so procured eyelets can work as an in vitro system for functional analysis. The surgical resection of human pancreatic tissue is the precondition for the isolation of human eyelets. We rely on patients undergoing a partial resection of the pancreas for specific pancreatic pathologies.
As a source of human pancreatic tissue, the surgeon cuts in the pancreas and performs the partial resection. Afterwards, the pancreatic fishermen is put on ice and carried to the Department of Pathology. The pathologist determines the amount of healthy appearing and soft pancreatic tissue to be procured.
For the eyelid isolation, at least two grams of pancreatic tissue are required for isolation. The tissue is placed in ucon solution and carried on ice to the laboratory. The first step in the eyelid isolation facility is to measure the weight of the pancreatic tissue.
The sample is then placed into a 10 centimeter dish. We already prepared one 500 milliliter flask with 150 milliliter of RPMI media and 1 250 milliliter flask with 130 milliliter of RPMI media. We then add the digestion enzyme solution containing one milliliter of DNAs and 20 milliliter of collagenase to the 250 milliliter flask and draw it up into a 10 milliliter syringe.
The cleaned pancreatic tissue is then distended by the injection of the enzyme containing media and minced into small pieces to increase the surface area and facilitate the process of digestion. This mincing step is crucial because the distention by injection alone does not allow a proper distribution of the enzyme within the tissue. In parallel, we assembled the digestion circuit.
As shown in this figure, the flow direction in the recorded chamber is in at the bottom and out at the top of the chamber. Afterwards, we are at the remaining enzyme containing media to the 500 milliliter flask up to a volume of 300 milliliter, and then insert the thermometer. The the recorded chamber contains a metal mesh with a poor diameter of 600 micrometer and free silicone nitride marbles with a diameter of 15 millimeter each.
We transfer the pancreatic tissue into the chamber, insert the mesh, close the chamber, and start the pump with a speed of 140 milliliter per minute. Once the temperature of the circuit achieves 37 degrees Celsius, we start to measure the time of digestion. The recorded chamber is shaken by hand according to the tissue composition.
To determine the optimal time point to stop the digestion, we take periodic samples, tisone stains, the separated eyelets red under the microscope. Once the eyelets appear to be separated from the aino tissue, we quickly stopped the digestion by putting the heating coil on ice and adding 200 milliliter of cold wash media to the circuit. Afterwards, we collect the eyelid solution by inserting the sample tube into 250 milliliter falcons until the circuit is empty.
The following washing procedure starts with the centrifugation step of 1000 RPM at four degree Celsius for five minutes. In the next step, we discard the snat Resus the eyelid solution in wash media and distribute it in equal fractions to the 50 milliliter falcons. The washing proceeds with another centrifugation step of 1000 RPM at four degrees Celsius for five minutes.
As an optional step, we distribute the palette into additional falcons beyond the original four, depending on the size of the palette. In order to improve the washing, again, we discard the sup natant and gently loosen the palate for eyelid separation from the aine tissue. The different tissue densities of the two parts enable the application of a fecal gradient.
First, we resuspend the pellet in fecal medium density 1.1 25 grams per milliliter. The pellet must be small enough to allow for homogeneous resuspension. Next, we slowly overlay this with the fecal media densities 1.0 81.0 60 and 1.0 37 with a volume of 10 milliliter each.
Afterwards, we centrifuge de gradients at 2, 400 RPM for 20 minutes at four degrees Celsius. Now you can distinguish the different components separated by their densities in the fractions. First, we discard the upper layer consisting of fat and connective tissue.
The fraction we now collect is the first layer of eyelid aggregate at 1.037 and 1.06, which mainly contains the isolated eyelets. For efficient isolation, it is important to collect the fractions separately. We then collect the fraction of the second eyelid aggregate at the interface of 1.0 60 and 1.0 80, which contains less pure eyelets than the first layer.
Next, we add the wash media to a total volume of 50 milliliter in order to dilute the fecal gradient and send to FUT at 1000 RPM for five minutes. At four degrees Celsius, we discard the SUP agent by suction because the palate might be very loose due to the cold stain. Thus, we have to repeat the washing step with eyelid culture media at 1000 RPM for five minutes at four degrees Celsius.
In the last step, we resuspend the human eyelets and eyelet culture, media and culture. The collected eyelets in the incubator at 37 degrees Celsius for 24 to 48 hours. After this culture step, the eyelids are ready for further processing.
This protocol should enable you to isolate human eyelids from pancreatic tissue after partial pancreatectomy. The rapid and cool transport of the pancreatic fishermen to the eyelid isolation facility is important to preserve the better cell viability. The isolation team has to keep in mind that the digestion process is a highly dynamic procedure.
The duration of the digestion process strongly depends on tissue composition. For example, the grade of fibrosis and the ability of sufficient enzyme injection into the pancreatic tissue. The individual adjustment of mechanical force within the recorded chamber and the length of the digestion process is crucial for good eyelid yield.
