The overall goal of this procedure is to isolate dendritic cells and epithelial cells from the human thymus. This is accomplished by first fragmenting the dissected tissue by mechanical disruption. Next, the tissue fragments are further enzymatically digested to produce a single cell suspension.
Finally, the cell suspension is enriched for the low density fraction of cells by perca separation. Ultimately, the dendritic and epithelial cells can be isolated from this fraction by magnetic or fact sorting, or a combination of both. The method we present here provides an efficient reproducible and cost-effective procedure for the successful isolation and subsequent study of rare cell population like dendritic cells and thymic epithelial cells found in the human thymus.
I rotta, a graduate student from our lab will demonstrate the procedure for you. Begin by placing the thymus tissue into a Petri dish containing sterile room temperature PBS, and rinse off any residual blood. Then add fresh PBS to the Petri dish and use forceps and scissors to clean away any blood clots, connective and fat tissue, and any parts of the tissue that do not appear to be healthy.
Once the thymus tissue is clean, weigh it for reference and then cut the tissue into uniformly sized one centimeter cubed pieces. Now cover the pieces with PBS and place them on ice. Then use the back of a 10 to 20 milliliter sterile syringe plunger to quickly apply mild pressure onto the tissue to remove the bulk of the thymocytes and to reduce the overall volume of the tissue to be digested.
The solution will become visibly cloudy. As the thymocytes are released, stir the dish gently and then use a glass aspirator to remove the thy containing supernatant ticking care not to accidentally aspirate the tissue pieces. It might be helpful to use a sterile cell culture scraper to concentrate all the tissue pieces to one side of the dish and then tilt the dish to a 45 degree angle and aspirate the remaining supernatant.
Rinse the dish with fresh PBS and continue to collect the supernatant until the PBS is relatively transparent. Perform a final wash with RPMI and then use sharp scissors to mince the tissue pieces as finely as possible, at least small enough to enter a five milliliter pipette to isolate dendritic cells or dc. First place up to 10 grams of mesh tissue pieces into 50 milliliter tubes with 10 milliliters of collagenase DNA solution and 10 milliliters of RPMI per five grams of tissue.
Then incubate the tissue suspension for 40 minutes under slow rotation in a 37 degrees Celsius thermal incubator. The enzyme solution will become cloudy as cells are released into it. At the end of the digestion centrifuge the cell suspension for two minutes at 110 G and room temperature to sediment the tissue fragments, collect the supernatant and then pellet the cell suspension.
This time discard the supernatant and resuspend the cells in 10 to 50 milliliters of complete RPMI. Depending on the size of the pellet, determine the number of viable cells by trian blue exclusion and then place the cell suspension at 37 degrees Celsius with a loose cap to isolate the thymic epithelial cells or t. Next Resus, suspend the tissue remnants in 10 milliliters of fresh collagenase DNA solution, plus 10 milliliters of RPMI and trypsin EDTA.
Further digest the tissue fragments for 40 minutes at 37 degrees Celsius with gentle rotation. During the last 10 to 15 minutes of the incubation, add fetal calf serum at a one to five ratio to neutralize the trypsin. After pelleting the tissue fragments, collect the supernatant and spin down the cell suspension again.
This time carefully aspirate and discard the supernatant, taking care to preserve the loose pellet. After counting the number of viable cells, place this cell suspension at 37 degrees Celsius as well. To enrich for low density fraction or LDF cells begin by pelleting the cell suspensions previously set aside for DC and tech enrichment while the cells are spinning down.
Set up a 1.07 gram per milliliter per call density gradient. According to this table, resus suspend both pellets up to one times 10 of the nine cells in six milliliters of the prepared per call solution. Mixing the cell solution sufficiently to get a homogenous suspension and then transfer the solutions to 50 milliliter Oakridge polycarbonate screw cap centrifuge tubes.
Next, use a pipette to slowly layer 30 milliliters of complete RPMI per tube on top of the perca cell suspension. Taking care not to mix the layers carefully transfer the tubes to a pre-cool centrifuge with a fixed angle rotor and separate the cells for 35 minutes. Then use sterile posterior pipettes to carefully transfer the enriched low density cell fractions from the interface between the per call and the medium into 50 milliliter tubes containing cold complete RPM.
I then fill the tubes with additional complete RPMI to dilute the remaining per call. Finally, wash the enriched cells two times incomplete RPMI then resuspend the pellet in fresh medium and determine the viable cell number In this first figure. A rather large nine centimeter piece of tissue obtained from a 6-year-old child is shown as just demonstrated.
An important first step is to clean the tissue of undesirable parts. As indicated by the white arrows, a single cell suspension of thymus tissue was then obtained and enriched for low density fraction cells. While a typical single cell suspension contains approximately 3%of H-L-A-D-R positive cells after density gradient separation, this percentage increases to 15 to 40%in the large sized cell.
Low density fraction in the high density fraction, which consists mainly of uniform. Small-sized thymocytes. There are virtually no such cells.Thymic.
Myeloid dendritic cells can be identified by the expression of a number of surface markers, including CD 11 C.This figure shows the efficient isolation of CD 11 C positive cells using a modified magnetic cell separation protocol after isolation. The purity of the CD 11 C positive DC was 93%as established by immunostaining of the isolated cells. On average, the recovery of isolated CD 11 C positive cells is five times 10 to the five to five times 10 to the six myeloid dendritic cells per 10 to the nine total thymic cells.
In this table, representative data obtained from several individual thymus samples of different age and tissue weight with a range of input single cell suspensions, as well as total low density fraction cell numbers and the subsequent CD 11 C positive yield and purity are shown in the antigen presenting cell enriched fraction only. Approximately 0.5%of the cells are CD 45, low EPCA positive or CD 45 negative EPAM positive cortical epithelial cells and medullary epithelial cells can be sorted from the trypsin digested CD 45 low negative enriched stromal cells as epam, low CDR two positive and epam high CD two negative respectively. The ululation of dendritic cells and thymic epithelial cell subsets from human ths.
It's time sensitive. The faster the ululation procedure, the better the condition of the cells. Once this protocol is mastered, dendritic cells can be isolated within five to six hours and thymic epithelial cells within eight to 10 hours.
After watching this video today, you should have a very good understanding of how to handle human thymus tissue for successful isolation of dendritic cells and thymic epithelial cells. The isolated cells can be then used in many different downstream applications for subsequent study to, in order to further characterize the cells as well as to understand their biology better.
