The overall goal of this procedure is to generate long-term cultures of adult mouse, spermatogonia, stem, and progenitor cells or SSCs. This is accomplished by first preparing tonically inactivated feeder cells. Next, a testicular cell suspension is prepared by enzymatic dissociation of adult testis tissue.
Then the testicular cells are plated on top of feeder cells to allow SSC colonies to form. The final step of the procedure is to manually pick putative SSC colonies for subculture in small culture wells. Ultimately long-term cell renewal and expansion of SSCs over many passages in vitro can be confirmed by immuno staining for broadly expressed markers of germ cells or more specifically of SSCs.
And by functional analysis using SSC transplantation in vivo. Generally individuals new to this method will struggle because the identification of small SSC colonies by phase microscopy is extremely challenging. Visual demonstration of this method is critical as the colony picking steps are difficult to learn because it may be difficult to see SSC colonies against a complex background of other cell types that are present in the earlier stages of the cultures.
To begin prepare feeder cells by culturing JK one cells in a 100 millimeter cell culture dish with 10 milliliters of filter sterilized feeder growth medium. When the culture reaches 95%confluence, split the cells in a one to six to one to 10 ratio by first washing the cells once with PBS containing no calcium or magnesium at Prewarm. Try trypsin EDTA and incubate at 37 degrees Celsius for five to 10 minutes with an equal volume of feeder growth.
Medium inactivate the trypsin, collect the cells and centrifuge at 300 times G for five minutes. Then resuspend cells in feeder growth. Medium preco multi-well cell culture plates by adding enough 0.4%gelatin solution to cover the well incubate for five minutes at room temperature.
Then remove any excess solution plate approximately 250 cells per square millimeter in the gelatin coated plates and incubate them at 37 degrees Celsius for 16 to 24 hours. Next, remove the culture medium from the wells. Then inactivate cell growth by adding a fresh solution of mitomycin C.Incubate at 37 degrees Celsius for four hours.
Next, remove the mitomycin C solution from the cells and wash them three times with DMEM. After removing the final DMEM wash, add enough stem cell medium to prevent desiccation of the feeders during packaging. Add SSCs the same day after harvesting adult mouse testes in a sterile fashion temporarily.
Store them in a covered Petri dish and immediately place on ice to prevent desiccation and maintain cell viability in a cell culture hood. Using sterile fine forceps and a fine scissor, make a transverse incision in the tunica abia without completely transecting the testes and use forceps to squeeze out the seminiferous tubules into a corner of the plate while keeping the plate on ice. Use fine spring scissors to rapidly mint the tubules for at least three minutes per pair of testes.
Collect the tubule fragments in a 50 milliliter conical tube and wash in approximately 40 milliliters of chilled 1%BSA in PBS centrifuge at 60 times G for 10 minutes. To separate tubule fragments from spermatozoa and debris, discard the supernatant for each pair of testes. Resus suspends the pellet in three milliliters of free warmed dissociation buffer.
Place the conical tube horizontally in a rack in a shaker set to 150 RPM at 37 degrees Celsius for 15 minutes. To maximize agitation. Spin down at 60 times G for 10 minutes to separate single cells from unassociated chunks.
Collect the supernatant in a separate 15 milliliter conical tube containing three milliliters of DMEM 10%FBS to neutralize the dissociation buffer. Place the tube on ice temporarily subject the unassociated chunks to another round of dissociation, followed by neutralization and combine the first tube with the second tube centrifuge for five minutes at 300 times. G resus.
Suspend the pellet in stem cell medium at a volume of 16 milliliters per testis plate. Two milliliters of cell suspension per well in a six well plate containing feeder cells. Mitotically inactivated with mitomycin C.Incubate at 37 degrees Celsius in 5%carbon dioxide after 48 hours.
Aspirate the medium. Add two milliliters of fresh medium and return the samples to the incubator. 48 hours later.
Add 0.5 milliliters of fresh stem cell medium to each well and return to the incubator following an additional 48 hours. Feed the cells three times per week as follows, until large discreet colonies of greater than 50 cells appear for routine feeding one and feeding.Two. Remove approximately 50%of the medium and add back an equivalent volume of fresh medium for the third.Feeding.
Aspirate all of the medium under a place with two millimeters of fresh medium. After replacing the medium with fresh stem cell medium passage, the cells by first using 10 x and 20 x objectives to identify the colonies with the microscope slightly unfocused. SSC colonies at the edge of the well will appear as homogenous bright clumps comprised of many 11 to 12 micron diameter cells in which it is difficult or impossible to discern the individual cell borders.
Since the cells appear fused together, non SSE colonies may appear darker, more granular or less homogenous with discernible borders. Using a 200 microliter pipette tip with the pipette man sent to 50 microliters, take up medium from the well and expel it to wet the tip. Then using the tip gently nudge the colony before rapidly withdrawing 50 microliters of medium to dislodge the colony by suction into the tip expel the medium containing the colony into one well of a 48 well plate containing inactivated feeder cells with 100 microliters of stem cell medium.
Add up to eight colonies to the same well without exceeding 500 microliters per well and prepare additional wells of the 48 well plate. With the passage one SSC colonies in seven to 14 days, or after large clumps of up to 500 have emerged, the wells will be ready to split to subculture cells that passage. One onto fresh faders.
Use a one milliliter pipette tip to tate the colonies of a semi confluent well by gently washing medium across the cells to loosen colonies while not disturbing unwanted feeder cells. Collect the triturated cells in a conical tube and centrifuge at 300 times G for five minutes. Aspirate the supernatant and resuspend the cells in fresh stem cell medium by thoroughly pipetting up and down to disrupt colonies.
Plate SSCs on freshly prepared feeder cells inactivated with mitomycin C as described earlier in this video, splitting them at a ratio of one to two for the first three passages and then one to four to one to six. Thereafter, use the three feeding per week schedule as described earlier. Plates should become confluent after seven to 10 days after five to seven passages.
Cultures should be comprised of a greater than 98%Germ cells shown here are passage. Zero wild type adult SSC colonies after seven days, three dimensional colonies are comprised of a layer of flat cells attached to the feeders or underlying extracellular matrix deposited by the feeders with multiple layers of SSCs growing on top. While healthy SSCs are brightly refract tile, and uniformly 11 to 12 microns in diameter, the cell borders are difficult to distinguish, and the size of the colonies may be highly variable, both within the well and between wells prepared from different mice.
Note that the morphology of the feeder cells will change gradually after switching from DMEM 10%to stem cell culture medium after inactivation with mitomycin C.Shown here are large colonies of greater than 50 cells that are manually transferred to a new well. The black structure is the pipette tip for comparison. Inactivated JK one feeder cells are shown here.
Without SSCs, large, tightly packed colonies will be present prior to routine subculture as shown in this image and maybe tightly or loosely anchored to the underlying surface. With practice, such colonies can be collected by tating or gently washing the plate without severely disrupting the feeder cells. Underneath the collected colonies can be broken up mechanically by pipetting up and down, or by Following this procedure.
Other methods like spermatogonia stem cell transplantation can be performed in order to answer additional questions such as what fraction of germ cells within a particular set of culture conditions represent authentic functional SSCs in vivo. After watching this video, you will have a good understanding of how to identify colonies visually and establish long-term cultures.I.
