Surgical Retrieval, Isolation and In vitro Expansion of Human Anterior Cruciate Ligament-derived Cells for Tissue Engineering Applications

The overall goal of this procedure is to develop a tissue engineered anterior cruciate ligament or ACL patch for a pair of partially torn ACL. This is accomplished by first obtaining the human ACL tissue from the patient and storing it in saline. The obtained ACL specimen is minced and digested with collagenase to obtain ACL derived cells.

The isolated cells are then cultured and expanded. The next step is to fabricate a two dimensional polylactic co glycolic acid scaffold. As a final step, the cells are then seeded onto this polymer film.

Ultimately scanning electron microscopy and immunofluorescence microscopy are used to show biocompatibility on the scaffold surface. Today we're here to demonstrate our technique in developing a patch for a partial ACL tears. The importance of this is the fact that ACLS do not heal on their own.

This is a new innovative technique that we've developed to help the ACL heal. To begin transfer the human ACL tissue received from pathology to a Petri dish with sterile saline and phosphate buffered saline or PBS mince the tissue using sterile scissors into one to two cubic millimeter pieces and wash the mince tissue with saline at least three times. Digest them in's tissue with 0.4%collagenase in complete supplemented ECCOs modified eagle medium nutrient mixture.

F 12 medium for four to six hours. At 37 degrees Celsius centrifuge the cells at 1000 cheese for five minutes. We suspend the cells in medium before washing the cells with medium two to three times, then seed in T 25 flasks and culture for two to three days.

Use a light microscope to visualize the cells cultured for two to three days In a T 25 flask. Maintain the cells at 37 degrees Celsius in the supplemented DMEM medium. Change the medium when cells show adherence and standard morphology as observed under a light microscope at day seven.

Wash the confluence cells with PBS, add trypsin and incubate the cells at 37 degrees Celsius for five minutes. Then add medium to the suspended cells to neutralize the trypsin before dividing the medium cell mixture between three T 25 flasks. Wash the confluence cells with PBS and add trypsin before Resus suspending them in freezing medium containing DMSO fetal bovine serum and the complete medium in a ratio of one to two to seven.

Store the cryogenic vials at minus 80 degrees Celsius. If the cells will be used within a month or in liquid nitrogen. If the cells will be stored for longer durations dissolve one gram of polylactic co glycolic acid or P-L-A-G-A in 12 milliliters of di chloro methane in a 20 milliliter scintillation vial and vortex the solution for eight hours at a constant speed of 800 RPM.

Transfer the dissolved solution to a glass petri dish lined with fluorinated protection paper. Then place the Petri dish under a vacuum hood for 30 minutes before leaving the Petri dish at minus 20 degrees Celsius overnight. Leave the plate at room temperature to ensure evaporation of residual solvent and obtain thin films of the polymer.

Next place the polymer films in a desiccate for 24 hours. Cut the polymer films into 12 millimeter diameter circular discs and store them in a desiccate until needed. After seeding the cells onto the controlled tissue culture, polystyrene or TCPS and the P-L-A-G-A scaffold at 50, 000 cells per disc.

Wash the scaffolds with PBS seven days subsequent to seeding. Use 1.5%glutaraldehyde in 0.1 molar caco dilate buffer to fix the cells overnight. Then add 2.5%osmium tetroxide in 0.1 molar caco dilate buffer for post fixation for one hour.

After washing the fixed cells with 0.1 molar caco dilate buffer, dry the fixed cells using serial ethanol dehydration for 15 minutes each. Further dry the cells in hexa methyl dilane overnight. Vent the chamber of the SEM sputter coating system with the button valve and raise the top plate.

Place the dried samples in the chamber and lower the top plate. Switch the pump control knob to start evacuating the chamber. Open the argon leak valve and wait for the vacuum to drop to 0.05.Millibar.

Coat the dried samples with a thin layer of gold palladium using a coating machine. Then return the argon leak valve to its closed position. Switch the control knob to off vent the chamber with the button valve and raise the top plate.

Remove the samples and store them in a desiccate for 24 hours. Finally, observe the samples under a scanning electron microscope for immunofluorescent staining. Also begin by washing the cells seated on the controlled TCPS and the P-L-A-G-A scaffold with PBS seven days subsequent to seeding.

Then fix the cells using cold, 70%ethanol for 10 minutes. Incubate the fixed cells at room temperature with 1%bovine serum albumin in PBS with 0.05%Triton X 100 for 20 minutes. Next, immerse the samples in 1%tween at room temperature for 20 minutes.

Add monoclonal mouse, anti beta actin antibody, and incubate the cells overnight at four degrees Celsius. The next day, wash the cells with 0.05%tween. Then add the secondary antibody to the cells and incubate for one hour at room temperature.

After washing the cells with PBS stain the cells with nuclear stain and mount using 80%glycerol. Finally, observe the cells under a confocal microscope. Human ACL derived cells were cultured and visualized under a light microscope.

The spindle or elongated shaped cells were seen growing on the surface of T 25 flasks after three days of culture, and a confluent monolayer was obtained by day seven. The presence of healthy viable cells indicated the successful retrieval and culture of the cells cellular attachment to the surface of polylactic, co glycolic acid scaffolds and the controlled tissue culture. Polystyrene was determined qualitatively via SEM adherence and proliferation of human ACL derived cells can be visualized here on each polymer surface.

Cellular confluence was also observed. Cellular morphology and adhesion analysis was determined via immunofluorescence staining. Green beta actin staining demonstrates that human ACL derived cells of both P-L-A-G-A and TCPS and display a normal non-stressed appearance.

After watching this video, you should have a pretty good understanding on how to design a tissue engineered patch for partial ACL tears using tissue engineering methods ACL cells derived from human ACL tissue, as well as studying the bowel compatibility with immunofluorescence and staining for this technique. Thank you very much.