Human Dupuytren's Ex Vivo Culture for the Study of Myofibroblasts and Extracellular Matrix Interactions

The overall goal of this procedure is to set up an ex vivo three-dimensional whole mount tissue culture system to study gene expression manipulation and its effect on extracellular matrix protein distribution. In this procedure, disease tissue is investigated from patients with dupy trons disease, a benign fiber proliferative disease that causes permanent flexion of the fingers due to the formation of nodules and cords in the palm of the hand. Following collection of nodules from diseased tissue, the fibrotic tissue is sectioned.

Then the ex vivo culture system is set up using the sectioned tissue. Next tissue parts are treated with different growth factors or chemical compounds. Alternative delivery of the compounds are performed by microinjection of adenoviruses and or lentiviruses to alter specific gene expression.

Ultimately, second harmonic generation and two photon microscopy is used for the assessment of extracellular matrix protein expression, and distribution. The major advantage of the three-dimensional culture over the two-dimensional culture of fibroblasts is that the tissue is maintained in its native extracellular matrix environment. The big advantage of this technique over two dimensional fibroblast culture is the rather large drug screen that can be done from patient derived material in nearly identical slices.

Moreover, this technique can be extended to the culture of hypertrophic scar, other connective tissue, and other skin models as well. Demonstrating the procedure will be Sophia Car Kauna, a PhD student from my laboratory. Begin this procedure with surgical collection of the nodule part of the cord from diseased fibrotic tissue as described in the text protocol Under laminar flow, open a sterile 12 well culture plate.

Place one culture insert in each well of the 12 well plate. Then fill the 12 well plate with 600 microliters of prewarm culture media by pipetting on the well and not directly on the membrane of the insert before placing the plate into the incubator. Next, transfer the nodule part of the cord from the 50 milliliter tube to a 100 millimeter Petri dish and add 10 milliliters of prewarm medium.

Ensure that the tissue is in contact with the liquid during the entire procedure. Remove the per nodular fat layer using meza bomb scissors and discard the fat tissue with the use of forceps and curved bladed scissors. Cut the tissue transversly into smaller pieces.

Transfer the 12 well plate from the incubator into the laminar hood. Check that the membrane of the insert has turned transparent and therefore is in contact with the medium. Using forceps.

Carefully lift a tissue part by touching the outer layer. Place one tissue part onto the membrane of the cell culture. Insert so that the tissue remains in the air medium interface culture, a maximum of two tissue parts on the same insert, and well ensure that the tissue is placed flat on the membrane in longitudinal contact with the medium such that the tissue neither floats off the membrane, nor is fully covered by medium.

Take care to avoid air bubbles. At this stage, it is possible to add growth factors or inhibitors of interest to the media. For example, test compounds A through E in replicates and or in a concentration curve include at least two control pieces of tissue under normal growth conditions to ensure that the experiment has been set up properly.

Incubate tissues for three to seven days in a tissue culture incubator. At this stage, if overexpression or knockdown experiments of a gene of interest are necessary, infect the tissue with either a lentiviral or adenoviral construct, employing a dissection microscope and an insulin syringe loaded with 50 microliters of phosphate buffered saline containing 10 microliters of the selected virus. Place the needle perpendicular to the center of the tissue.

Slowly inject the content of the syringe without retracting the needle. When puncturing the tissue, do not let the needle go through the other side, but maintain the needle within the tissue itself. Following injection, transfer the tissue back to the 12.

Well plate. Close the culture plate and place it into the incubator. Remove transwell plates from the incubator and place a single tissue part in a 35 millimeter cell culture plate containing 500 microliters of medium.

Position the tissue so that the long axis is flat on the plate, keeping the tissue wet at all times, but not floating. Then place the water immersed objective of the multi photon microscope directly contact with the tissue. Obtain images with an excitation wavelength of 800 nanometers and collect emitted light from 371 to 425 nanometers for collagen and 474 to 532 nanometers.

For elastin. Perform two photon microscopy in an upright confocal microscope, equipped with a femtosecond laser and a 20 x water immersion objective with a 1.0 numerical aperture processed confocal stacks using the manufacturer's software. Shown here are representative results of cultured duy trons tissue parts subjected to control conditions incubation with compound A or incubation with compound B.The effect of anti or pro fibrotic potential of compounds is evaluated in tissue sections by immunofluorescence.

For alpha smooth muscle actin, a protein expressed by myofibroblasts shown here in green animated imaging of three three-dimensional myofibroblast distribution is visualized through alpha smooth muscle actin whole mount immunofluorescence shown here in red. This figure demonstrates proof of principle of ex vivo viral mediated gene expression, modulation in whole mount tissue, adenovirus expressing red fluorescent protein and lentivirus expressing green fluorescent protein were used to assess cellular uptake. Evaluation of viral delivery was performed 24 hours after microinjection by confocal microscopy after tissue fixation in 4%PFA and preparation of 0.7 micron cryo sections finally shown here are representative results of multi photon microscopy, imaging of endogenous collagen and a elastin distribution in whole mount tissue After its development.

This technique has paved the way for researchers in the field of fibrosis to study extracellular matrix protein in relation to other cell types present in the human tissue. Following this procedure, it is possible to analyze gene expression and protein profiling by second harmonic generation QPCR, fax analysis and multi photo imaging.