The overall goal of the following experiment is to develop an easy and simple method to produce uniform, three dimensional neuroendocrine tumor steroids, and a simple method to process the HistoGel embedding for studying the histology of these steroids. This is achieved by evenly coating 24 well plates with aeros on which human neuroendocrine tumor cells in a single cell suspension are seeded. The plate is then incubated on an orbital shaker in the incubator overnight at a speed lower than 40 RPM, and then transferred to a stable shelf in the incubator.
Spheroid will form by day three or four, and their growth and integrity are monitored by phase contrast microscopy in order to study their histology. The 3D steroid are embedded in warmed HistoGel prior to routine paraffin processing results are obtained that show growth of neuroendocrine tumor 3D steroids based on images taken during phase contrast Microscopy. Histology of the 3D steroid is analyzed by h and e and immunohistochemical staining of paraffin block sections of the 3D steroids.
The main advantage of this technique over the existing method is that it's simple, easy, reliable, and cost effective. This method can facilitate research in the drug discovery field by helping to quickly identify therapeutic drugs for the neuroendocrine tumor patients. In general, individuals new to this method will struggle initially at least to grow uniformly shaped in size 3D steroids.
The key to this is to ensure that your 24 well plates are evenly coated with agros and also to ensure that your single cell suspensions are well dispersed and are accurate. To understand the histology of the 3D steroids, we need to perform paren processing and immunochemical staining. Histo embedding of the 3D steroids is the key step which will be demonstrated by late on today To prepare 1%augurs for coating 24 well plates add one gram aeros to 100 milliliters of deionized water in a 250 to 500 milliliter bottle and autoclave to sterilize the aeros after sterilization.
Transfer the bottle into a 70 degree Celsius water bath for about an hour. For the aeros to cool to 70 degrees Celsius, the aeros must be kept sterile at all times. In a biosafety cabinet, use a repeater pipette to dispense 200 microliters of aeros into each well of 2 24.
Well flat bottom plates for this protocol. Any kind of 24 well flat bottom plates would work as long as the plates can be imaged under a phase contrast microscope swirl the aeros around the well to make it cover the well evenly. 200 microliters of aeros is optimal for forming a concave surface to allow spheroid to form consistent spherical shapes.
After about 10 minutes, the aeros coated 24 well plates are ready to be used. The bon one cells used in this experiment are human pancreatic neuroendocrine tumor or NET cells maintained cells in D-M-E-M-F 12 with 10%FBS medium in a sterile tissue culture dish in a 37 degree Celsius humidified incubator with 5%carbon dioxide. When monolayer bond one cells reach 70 to 80%Co fluency, single cell suspensions are prepared in a biosafety cabinet.
Remove the medium and wash cells with PBS twice dislodge cells from the dish by adding one milliliter trip le and incubating at 37 degrees Celsius for five minutes. Check under the microscope to confirm that all cells are detached from the dish and then add nine milliliters. Growth medium to the trypsin.
Eye cells pipette cells up and down a few times to ensure an even single cell suspension. Use a 70 micron cell strainer to filter the cells. Collect filtered cells after performing a cell count, prepare a suspension of 5, 000 cells per milliliter in D-M-E-M-F 12 with 10%FBS growth.Medium.
The number of cells to be plate is should be determined empirically based on the size of the spheres on day six. Ideally, you'd like to have a spirit size between 300 to 400 nano micrometer and diameter. For this reason, we plate a thousand cells per 200 microliters per well.
To begin this procedure, overlay 200 microliters of the single cell suspension onto each aeros coated well of the previously prepared 24 well plates seal the 24 well plates with sterile ceiling tape to prevent evaporation. Place the aero coated 24 well plates containing bon one cells on an orbital shaker at a very slow agitation in a 37 degree Celsius humidified incubator with 5%carbon dioxide in air, incubate overnight on the following morning, move the plates to a stable platform and keep the cultures growing in the incubator. Every three days.
Add 200 microliters of growth medium to each well of the plates. To avoid disturbing the phe, touch the pipette tip to the side of the well and let the medium flow down the wall into the well monitor OID formation in the 24 well plates under a microscope. Drug treatment of the 3D steroids is initiated at day six of culture.
When spheroids are around 300 to 400 microns in diameter, this is denoted as day zero for drug treatment at day six. Each well of the plates contains about 400 microliters of medium to ensure that the drug treatments are at a one x final concentration in each well for each treatment dose make three times the desired concentration, dilute the appropriate amount of drug stock in five milliliters of growth medium in a 15 milliliter conical tube, which is enough for eight replicates the PHE in each row of the 24 well plates are characterized as vehicle dose 1, 2, 3, 4, and five. There are four replicates for each dose of treatment on each 24 well plate.
Usually 3D phe are treated in at least 2 24 well plates resulting in at least eight replicates for each dose of treatment. Before adding the treatment to each, well spin down the 24 well plates to make sure that all the condensation on the ceiling tape goes into the well due to the floating feature of the 3D steroid. The medium in each well is not removed after centrifugation.
To avoid disturbance of the sphe, carefully add 200 microliters of each dose of treatment into the appropriate wells along the wall of each. Well seal the 24 well plates with ceiling tape and incubate the cultures in a 37 degree Celsius humidified incubator with 5%carbon dioxide in air. Monitor the 3D steroid cultures at each chosen time point after drug treatment with a phase contrast microscope.
A five x objective is ideal because the PHE quickly outgrow the imaging field with a 10 x objective. Acquire images of the steroid in each well at each time point prior to HistoGel. Embedding the net 3D steroids are collected from the 24 well plates and fixed in 10%Formin.
Place a tube of cold HistoGel into a beaker filled with water. Heat the beaker in a microwave for one minute or until the gel is completely liquified. Keep the tube in the same water filled beaker for the duration of the procedure.
Using a disposable plastic pipette. Transfer all the steroid into a biopsy cryo mold. Let the steroid sit for a minute to sink down to the bottom of the biopsy cryo mold.
Try to get rid of the liquid in the biopsy cryo mold with Kim wipes. Very gently push the steroid to the center at the bottom of the biopsy cryo mold using a toothpick. Next while using the Pasteur pipette to very gently maintain the steroid at the center of the biopsy cryo mold.
Add a thin layer of warmed HistoGel to the biopsy cryo mold. Don't add too much HistoGel. Just enough for stabilizing the steroids at the center.
Let the steroid in HistoGel sit for one to two minutes at room temperature or until the HistoGel is solidified. Fill the rest of the biopsy cryo mold with warmed HistoGel and allow it to solidify room temperature for about 10 minutes. Just prior to popping this OIDs HistoGel block out from the biopsy cryo mold.
Leave the cryo mold on ice for one minute, then pop out the intact steroid HistoGel block. Wrap the block in a piece of bio wrapp and put it in a tissue and biopsy cassette. Store the tissue and biopsy cassette in a container with 70%ethanol.
Subsequently, routine tissue processing procedures for paraffin embedding are performed when grown on aeros coded 24 well plates. Different net cell lines show different morphologies as seen here. The human pancreatic net cell line bond one and human lung net cell line H 7 27 formed 3D steroids.
In contrast, qgp one. Human pancreatic net cells show large grape like porous masses, which are not regarded as steroids. After seeding bond, one cells on the agros coated 24 well plate multicellular steroid formation typically occurs by the third or fourth day, and the steroid become rounder by the sixth day.
Due to the nutrient and oxygen limitation. Cells in the dense core of the steroid start to die around day 10 and by day 17, the steroids begin to disintegrate. Due to the large size or in some cases the ejection of the necrotic cord, the steroid typically can grow up to 1000 microns in diameter.
In this experiment, day six 3D steroid were chosen as the start point for treatment with the histone decease inhibitor trikes statin A or TSA. It has previously been reported that histone deacetylase inhibitors exhibit antiproliferative and proto effects on net cells. These images track the morphology of the 3D steroids at the start of treatment and at 24 48, 72 and 96 hours later.
V indicates treatment with the vehicle, which is DMSO in this case. D one to D five represent treatment with increasing doses of TSA the size of each individual's. OID was estimated automatically by a custom developed MATLAB computer program.
At least eight sphs were measured per dose of TSA treatment at each time point, and the results are plotted in this graph relative to the vehicle. All doses of TSA showed some degree of growth inhibition on 3D OIDs. At TSA concentrations of 125 nano molar and 250 nano molar growth of 3D steroids was strongly suppressed to understand the histology of the net 3D steroid.
H and e staining and immunohistochemical staining were performed on 3D steroid that had been cultured for 17 days. KI 67 is a marker for proliferating cells while cleaved caspase three is a marker for apoptotic cells. The results indicate that the outer layer cells of the 3D steroid are actively proliferating, but the cells within the core are mostly necrotic or apoptotic.
Following this procedure, additional assays such as the cell titer glow assay can be performed in order to determine these cell viability of these 3D steroids. After its development, this technique can be extended to 96 wall plates to perform high throughput screens, which will facilitate therapeutic drug discovery in cancer research.
