Generating primary cellular models or organoid-like structures is key to answering many contemporary questions in biology. For example, we've been interested for quite some time in the mechanisms used by human viruses to facilitate replication in the salivary gland as it is an important site for horizontal transmission. The development of the salisphere-based system described in this protocol is a crucial advancement in our quest to answer these types of questions.
This technique allows us to generate primary cells from a variety of different patient samples. The cellular models are more reminiscent of the in vivo type situation as the cells are not modeled or transformed like many cell types commonly used for in vitro experiments. We've used this protocol to generate similar cells from mouse primary submandibular gland and the protocol would likely be useful for other organs with similar cellular structures.
Within two to four hours after removal from the patient, on a culture plate, use autoclaved sterilized dissecting scissors and surgical forceps to mince the salivary gland tissue into fine pieces at the size of one to two millimeters. Add six milliliters of the Dispase/Collagenase solution to the minced tissue. Then incubate at 37 degrees Celsius for approximately 30 minutes to one hour.
Disrupt the tissue by pipetting with a five milliliter serological pipette 15 to 20 times. Incubate at 37 degrees Celsius for another 30 minutes to one hour. Repeat the pipetting and incubation step two to three more times or until the tissue resembles a slurry and can easily pass through the pipette opening.
To coat wells with BMM, slowly pipette 500 microliters of overnight thawed BMM into each well of a six well plate. Slowly swirl the plate to evenly distribute the BMM over the wells. Then incubate the coated plates at 37 degrees Celsius for at least 15 minutes prior to use to allow the BMM to solidify.
First, transfer the Dispase/Collagenase solution containing homogenized tissue to a 15 milliliter conical tube. Wash the wall of the plate once with six milliliters of DPBS to transfer the remaining cells into the conical tube. Then through a 70 micrometer nylon mesh cell strainer, filter the homogenate into a new 50 milliliter conical tube to remove undigested tissue.
Centrifuge strained cells for five minutes at 500 times g. Aspirate the supernatant. Then resuspend the cell pellet in 10 milliliters of 1X RBC lysis buffer.
Incubate for five minutes at 37 degrees Celsius. Then add 20 to 25 milliliters of DPBS to neutralize the RBC lysis buffer and minimize lysis of salivary cells. Centrifuge for five minutes at 500 times g.
White cell pellet indicates complete lysis of all RBC. If the pellet has red color, repeat treatment with RBC lysis buffer. Aspirate the supernatant and resuspend the pellet in one to two milliliters of BEGM and then transfer the resuspended cells onto BMM coated wells.
Incubate at 37 degrees Celsius. Once plated on BMM, cells will form into spherical structures or salispheres over a two to three-day period. Cells can be maintained as salispheres for about five to seven days before the BMM begins to degrade allowing the cells to access and adhere to the plastic and grow as a monolayer.
First, aspirate the media from the wells and add one milliliter of Dispase/Collagenase solution to each well. Incubate at 37 degrees Celsius for approximately 15 minutes or until BMM has mostly dissolved. Then transfer the cells to a 15 milliliter conical tube and wash wells once with one to three milliliters of DPBS to obtain the remaining cells.
Centrifuge for five minutes at 500 times g. Aspirate the supernatant and resuspend the pellet in two milliliters of Trypsin. Incubate again at 37 degrees Celsius for 15 minutes.
Next, add in to the tube any complete media containing 10%serum to neutralize the Trypsin. Centrifuge for five minutes at 500 times g. Aspirate the supernatant to remove residual media, Trypsin and serum and then resuspend the cells in DPBS.
Centrifuge for five minutes at 500 times g. Aspirate the supernatant and resuspend the pellet in BEGM. To maintain the pellet as spheres, plate the resuspended cells onto solidified BMM coated culture dishes.
Culture all cells in a humidified incubator maintained at 37 degrees Celsius with 5%carbon dioxide for five to seven days prior to subculturing. Feed with fresh BEGM every two to three days. To proliferate the cells as a monolayer onto plastic culture dishes, aspirate the BEGM media from the dish and wash once with DBPS to remove residual media.
Add two milliliters of Trypsin and incubate at 37 degrees Celsius for 15 minutes or until cells have fully detached. Neutralize Trypsin using four milliliters of any complete media containing 10%serum. Then gently tilt the dish and transfer cells to a 15 milliliter conical tube.
Wash the plate once with approximately five milliliters of DPBS to collect the remaining cells. Centrifuge for five minutes at 500 times g. Aspirate the supernatant.
To remove residual media, resuspend cells in six to 10 milliliters of DPBS. Centrifuge for five minutes at 500 times g. Aspirate the supernatant and resuspend in BEGM.
Then plate the cells onto the treated plastic tissue culture dishes. Culture the cells in a humidified incubator maintained at 37 degrees Celsius with 5%carbon dioxide for five to seven days prior to subculturing. Feed with fresh BEGM every two to three days.
In this protocol, after plating cells from the digested tissue onto BMM for two to three days, cells readily formed small clusters that continued to expand in size up to 15 to 20 cells per cluster. Salisphere cells were plated onto cell cultured treated plastic and grown as a monolayer where they exhibit morphology consistent with cells of epithelial origin. Proper aseptic technique and care is needed to avoid bacterial or fungal contamination.
No amount of antibiotics or antimycotics will save a culture swarming with microbes. We are answering questions about viral pathogenesis and transmission. But for others, this provides a way to study human salivary cells for any number of applications such as tissue regeneration therapies.
For us who study human Cytomegalovirus, this technique has allowed us to begin exploring the molecular mechanisms the virus utilizes to infect and ultimately transmit to new hosts. Nothing used in this protocol is inherently hazardous but caution should always be taken when handling human tissue and any sort of chemical reagents.
