Tissue Collection of Bats for -Omics Analyses and Primary Cell Culture

In this video, we provide a step by step guide to collecting bats in a humane way, intended to minimize impact on populations and maximize scientific value. The advantages of our approach are that we maximize the amount of potential molecular and morphological data for each bat, preserve tissues to retain their future value, and minimize harvesting wild individuals. Dissection protocols are difficult to verbally demonstrate.

Many of the tissue preparation techniques we present here are best communicated through visualizing how different organs are identified, and then properly dissected and stored. Prepare all vials before starting any dissections to avoid delays. For RNA, set aside the number of vials needed for each specimen.

Label each tube with the tissue type that will be collected, as well as standard specimen identification information. Fill the vials 50%full of RNA stabilizing solution, and chill to four degrees celsius. Take care not to fill the vials completely with RNA stabilizing solution, as the tube may explode when placing it in liquid nitrogen.

Immediately after euthanasia, perform decapitation of the bat specimen as described in the text protocol. Skin the skull from the hair, fascia, and skull muscles, including the skin on the nose. Take care not to break the front end of the nose.

Remove the eyes with forceps, using strong enough force to detach the optic nerve. Place the eyes in a two milliliter vial of RNA stabilizing solution. Use scissors to make a sagittal cut on the dorsal portion of the skull starting at the neck, taking care not to damage the brain.

Then, use forceps to gently pull back both sides of the skull, until it is cracked open to expose the brain. Ensure that the skull of the cranial end has been removed, so that forceps can easily scrape away the brain. Gently scrape the brain with forceps.

The brain will be very soft. The olfactory bulb will become visible, sitting at the ventral portion of the interior of the skull. Try to keep the olfactory bulb attached.

If possible, keep the brain shape intact, and immediately place it on dry ice, or in a five milliliter vial of RNA stabilizing solution. On the caudal end of the skull, the cochlei should now be laterally visible on each side of the head. Use forceps to gently pull the cochlei.

Then, put them in one two milliliter vial of RNA stabilizing solution. Make two incisions where the top and bottom jaw join, and remove the mandible. The cribriform plate will become apparent.

This is a critical bone to keep the researcher oriented. It can be identified as the most anterior region of the skull, with multiple foramen, and with two grooves where the olfactory bulb rests. Once the mandible has been removed, remove the rostrum from the remaining part of the skull.

Ensure that the jaw includes the cribriform plate. Place the nose in RNA stabilizing solution, and store at four degrees celsius overnight. From the lower mandible, cut the tongue with scissors, and place in a two milliliter vial of RNA stabilizing solution.

Place the nose vial in liquid nitrogen following the overnight soak in RNA stabilizing solution. After 24 hours of soaking on ice, or in the fridge, place the samples in liquid nitrogen. Use a scalpel to pierce through the abdominal cavity, making a longitudinal incision up to the ribs.

This forfeits the skeletal frame. Strip the skin to reveal the pectoral muscle, and take at least two samples of muscle. Tissues for both RNA and DNA are collected during these dissections.

Place tissues for high molecular weight DNA into dry cryovials, and flash freeze in liquid nitrogen. Place tissues for RNA in RNA stabilizing solution. Cut through the sternum, and pull away the ribs.

Now, retrieve the lung and heart. Use the scalpel and forceps to separate the heart from the lung. Use a scalpel to break up the lung, and place in RNA stabilizing solution.

Collect the heart, which can be taken whole, but should be sectioned into halves, so that the RNA stabilizing solution soaks thoroughly. Now, take samples from the liver. Take at least two samples of liver.

Place one sample into a vial to remain frozen for high molecular weight DNA, and place the other sample in RNA stabilizing solution. Follow the hepatic duct to find the pancreas and gall bladder. Transfer the gall bladder to a vial of RNA stabilizing solution.

Find the stomach, and the feather-like spleen at it’s base, appearing as a different shade of purple. The pancreas should also be visible here as a weight structure. Collect the stomach, the spleen, and the pancreas, in respective vials of RNA stabilizing solution.

Collect small samples of the small and large intestine. Place in respective vials of RNA stabilizing solution. Take one of the kidneys, and follow their ducts to the bladder, then place in respective vials of RNA stabilizing solution.

Use the other kidney as a guide to find the testes if male, or uterus and ovaries if female. Collect one or both gonads if possible. Also take samples of various parts of the skin of the wing, and keep in separate vials.

The main advantage of our approach is that it allows us to sample animals in a non-lethal manner, while still generating enough material for genomic studies, as well as functional molecular studies. Our protocol allows the preparation of primary fibroblasts from wing tissue. The cells represent a precious renewable source of genetic material, as well as a model for functional and molecular explorations in bats.

Wing clips that have been preserved in growth media as per section six of the written protocol should be transferred to the tissue culture facility. Transfer the contents of the tube into a 15 milliliter conical centrifuge tube. The wing membrane biopsy is used for this demonstration.

Carefully remove the growth medium. Gently wash the biopsy two times with 500 microliters of sterile PBS. Add 500 microliters of one milligram per milliliter collagenase four to the tube.

This will cause digestion of the tissue into individual cells. Incubate overnight at 37 degrees celsius without agitation. On day two, make a fresh growth medium, and prewarm it to 37 degrees celsius.

Prepare a six well tissue culture plate by adding two milliliters of fresh prewarmed growth medium in each well of the plate to be used. Store this plate in a 37 degrees celsius and 5%carbon dioxide incubator until needed. Remove the 15 milliliter tube containing the cells from the incubator, and quench the digestion reaction by adding one milliliter of fresh growth medium.

Resuspend the cells by carefully triturating the solution with a P1000 pipette tip to achieve a single cell suspension. Gently spin down the cells in a tabletop centrifuge for three minutes at 300 times G.Discard the supernatant by gently removing 80 to 90%of the liquid with a P1000 pipette. Resuspend the pellet in 500 microliters of prewarmed growth medium.

Gently triturate the suspension to ensure that the pellet is no longer visible, and that cells are sufficiently suspended, making sure to avoid small pieces of tissue which may still be visible on the walls. Gently pipette the entire volume of cell suspension into a single well of the six well plate that was prepared earlier, containing the prewarm growth media. Gently rock the plate from side to side and front to back two to three times to help cells distribute over the well surface in a single layer.

Check the plated cells under a microscope. They should be single cells that appear balled up and floating, but very dense. Carefully place the plate into an incubator preset to 37 degrees celsius and 5%carbon dioxide.

After approximately 24 hours, observe the cells under the microscope to determine the health of the culture. Cells should now be attached to the plate surface, and appear flattened. Maintain the cells in a humidified incubator preset to 37 degrees celsius and 5%carbon dioxide.

Cells should be observed under the microscope regularly to determine the need for media refreshment or splitting. Whenever necessary, carefully aspirate approximately 50%of the medium from the well, and gently add one milliliter of prewarmed growth medium to the side of the well, so as not to disturb the cells. When it is necessary to passage the cells, carefully aspirate approximately 90%of the growth medium.

Wash the cells very gently by adding one milliliter of sterile PBS to the wall of the well, so as not to disturb the cells. Gently rock the plate back and forth and side to side two to three times. Carefully aspirate all of the PBS from the plate before washing the cells again.

Add trypsin EDTA to the well, and gently rock the plate to cover the whole surface of the well with trypsin EDTA, and incubate for one and a half minutes at room temperature. Quench the reaction by adding one milliliter of fresh prewarmed growth medium. Pipette up and down approximately five times to wash the cells from the surface of the plate, and ensure the cells are in suspension.

Place the cell suspension in a 15 milliliter tube, and spin down the cells in a tabletop centrifuge for three minutes at 300 times G.Discard the supernatant by gently removing 80 to 90 percent of the liquid with a P1000 pipette. Resuspend the pellet in one milliliter of prewarmed growth medium, and gently triturate the suspension. Ensure that the pellet or large fragments of the pellet are no longer visible, and cells are in a single cell suspension.

To count the cell using an automated cell counter, mix a ten microliter aliquot of the suspended cells one to one with trypan blue to detect viable cells. Incubate for one minute, and pipette 10 microliters of the solution onto the counting slide. Insert the counting slide into an automated cell counter to count the cells using the appropriate settings.

Prepare freezing media by combining growth medium with DMSO to obtain a final concentration of 10%DMSO. The pellet should be prepared from an 80 to 90%confluent well of a six well plate. Resuspend the pellet in one and a half milliliters of freezing medium.

Place approximately 750 microliters of cell suspension in each of two separate cryovials. Place the vials in a cryogenic freezing container, so that the cells freeze slowly to maintain cell vitality. Immediately place them at minus 80 degrees celsius.

To thaw the frozen cells, prepare a six well plate containing two milliliters of prewarmed growth medium in each well that will be used. Maintain the plate in the 37 degrees celsius and 5%carbon dioxide incubator until needed. Take one vial of cells from liquid nitrogen, and place it in a warm water bath to rapidly thaw the cells.

This should take two to three minutes. As soon as the solution in the vial has thawed, gently pipette up and down to homogenize the solution, and immediately place the entire solution in one well of the six well plate. Gently rock the plate from side to side and front to back two to three times to help cells distribute over the well surface in a single layer.

Place the cells in the incubator at 37 degrees celsius and five percent carbon dioxide for 24 to 48 hours. Monitor and passage the cells as demonstrated. Representative results of DNA extractions are shown for standard DNA analyses from three bat species.

A single large band indicates minimal fragmentation, and similar sized fragments of DNA from each respective extraction. A common indicator of success for RNA extractions is the RNA integrity number, or RIN, with values above eight representing high quality in tact RNA. Shown here are RIN values of RNA extractions from 13 different tissue types of neotropical bat species, sampled at four different localities.

Tissues sampled from species in the Andes, Costa Rica, and the Dominican Republic were placed directly in liquid nitrogen after soaking in RNA stabilizing solution at four degrees celsius overnight. Tissues sampled from species in the Amazon were placed in liquid nitrogen approximately one week after placing an RNA stabilizing solution, and kept at four degrees celsius continuously. Even in such cases, RIN values were comparable to those immediately placed in RNA stabilizing solution, and directly into liquid nitrogen.

Following tissue culture, bat cell cultures should cover 80 to 90%of the plate surface, and maintain their original morphology. This represents the optimal stage for splitting. After more than six passages, cells will change their morphology to become larger and longer, and the cells will enter senescence.

Bright balled up cells represent dead cells. Cells in culture represent a renewable source of material, like DNA, RNA, and protein. These cell lines can be frozen, thus providing a resource that can be used over many years of research.

This allows genomic, transcriptomic, and functional studies that will be challenging using primary tissues. For example, cells can be modified genetically or chemically to observe effects on cellular phenotypes.