In Vitro ExperimentsTissue Harvesting

Six weeks before the in vivo experiments began, a 2-mm-diameter tissue sample was harvested from the ear of a 4-month-old longhorn-crossbreed calf weighing 70 kg (4B Livestock, Midway, Texas) by punch biopsy with a trephine (Nasco, Fort Atkinson, WI) (Figure 1). The biopsy was performed under sterile conditions and local anesthesia. The tissue sample was immediately placed in sterile cell culture medium containing antibiotics (Table 1) and transferred to a sterile hood. The sample was kept at 4ºC for a minimal amount of time to keep the tissue viable.

FIGURE 1 Harvest of bovine auricular cartilage from the ear of the tissue-donor calf. (Left) A 2-mm-diameter biopsy sample (arrow) of auricular cartilage being held by a pickup. The trephine used to obtain the sample is shown still in the ear. (Right) Biopsy site (arrow) at the ear after the trephine was removed.

Tissue Isolation

Under the sterile hood, auricular elastic cartilage was removed from the surrounding dermal tissue of the biopsy sample by microdissection. The isolated cartilage was placed in 10- x 10-mm tissue culture dishes with 2% antimycotic-antibiotic phosphate-buffered saline (Table 2) and incubated at 37ºC for 10 minutes. Multiple small pieces of the cartilage were placed in 6-well tissue culture plates in 500 μl of modified RPMI cell culture medium and incubated at 37ºC.

*Equitech-Bio, Inc., Kerrville, TX.
†100 mM MEM sodium pyruvate solution; GIBCO-BRL, Rockville, MD.
‡Sigma, St. Louis, MO.
RPMI 1640 medium (90%)

Fetal bovine serum (10%)*

Sodium pyruvate (1X) †

Minimum essential amino acids (500 μl)

L-Glutamine (2mmol/L)

Penicillin G

Streptomycin

HEPES (1X)

Table 1. Composition of Modified RPMI 1640 Cell Culture Medium

*Sigma, St. Louis, MO.
Dulbecco’s phospate-buffered saline (1X)*

Penicillin G sodium (10,000 U/ml)

Streptomycin sulfate (10,000 μg/ml)

Amphotericin B (25 μg/ml)

Table 2. Composition of 2% Antimycotic-Antibiotic Phosphate-Buffered Saline
Solution

Cell Culture

Chondrocytes growing from the auricular cartilage were cultured under sterile conditions and maintained in a humidified 5% CO₂ incubator at 37ºC. Every 12 hours, drops of medium were added to each culture well. After 5-7 days, when chondrocytes became adherent to the culture dishes, cartilage pieces were removed. The chondrocytes were then passaged as follows. First, they were trypsinized in a solution of 0.5 g trypsin and 0.2 g EDTA (Sigma, St. Louis, MO). Then, when the cells became detached, fetal bovine serum was added to inactivate the trypsin. The resulting single cell suspension was then transferred to tissue culture plates to establish a monolayer of chondrocytes. Thereafter, the cell culture medium was changed every 48 hours. Chondrocytes were passaged twice, and cells of the second passage were used for subsequent experiments.

Histology

Fine (1-μm-thick), paraffin-embedded sections of pure elastic cartilage tissue were stained with Verhoff-van Gieson stain to visualize elastic fibers, Masson’s trichrome stain to visualize collagen fibers, and hematoxylin and eosin to assess tissue and cell morphology.

Immunocytochemistry

A portion of the second-passage chondrocytes from each culture plate was plated on slides and fixed in 1% formalin forimmunocytochemistry using a double-antibody labeling technique. Paraffin-embedded sections of pure elastic cartilage tissue immunostained in the same way served as a control. The primary antibody used was collagen type II (NCL-Coll-Iip; Novocastra); the secondary antibody used was a biotinylated immunoglobulin specific for the primary antibody (Vectastain^® Elite IgG; Vector Laboratories, Burlington, CA). In brief, deparaffinized and hydrated cartilage tissue sections and cells were incubated in 3% hydrogen peroxide for 10 minutes to quench endogenous peroxidase activity. All specimens were then incubated with normal mouse serumfor 20 minutes to block nonspecific binding sites. Next, all specimens were exposed to a 1:50 diluted solution of the primary antibody for 1 hour. After repeated washes, all specimens were incubated with the secondary antibody for 30 minutes. Next, all specimens were exposed to avidin DH and biotinylated enzyme (Vectastain^® Elite ABC reagent) for 30 minutes and then to diaminobenzidine as a peroxidase substrate for 30 seconds. Finally, all specimens were counterstained with hematoxylin for 1 minute. Control incubations were performed in the absence of the primary antibody.

Cell Seeding
Four implantable pneumatic LVAD’s (HeartMate®; Thermo Cardiosystems, Inc., Woburn, MA) were used for the in vitro cell seeding experiments. The HeartMate® has two luminal artificial surfaces: a flexible diaphragm made of a “biomer” (i.e., polyurethane flocked with polyester microfibrils) and a metal housing made of sintered titanium microspheres⁶ .One of the four LVAD’s used in the in vitro experiments was later used in the in vivo experiments described below.

Seven days before the in vivo experiments began, each of the four LVAD’s was seeded with a total of 3 x 10⁷autologous cells under sterile conditions. The inlet and outlet conduits of each LVAD were closed, and the LVAD was placed in a humidified 5% CO₂ incubator at 37ºC. Each LVAD was tilted 15 degrees every 2 hours for 24 hours during cell seeding to ensure complete cell coverage of surfaces. After seeding was completed, three samples of cell culture medium were collected from each LVAD and assessed for the number of nonadherent cells using a Coulter counter (Coulter, Hialeah, FL). The seeding procedure and the consecutive assessment of seeding efficiency were repeated to ensure complete cell coverage of both luminal surfaces of the LVAD. Each LVAD was then incubated for 4 days in the same incubator. Cell culture medium supplemented with 50 μg/ml of sodium ascorbate (Sigma, St. Louis, MO) was changed every 48 hours to promote extracellular matrix synthesis in order to maximize the adherence of cells to both artificial surfaces. After each medium change, samples were collected to assess the number of nonadherent cells as before and to calculate seeding efficiency based on the number of initially seeded cells.
Cell Preconditioning
Because preconditioning a.) Promotes good cell adherence once an LVAD is implanted and perfusion initiated and b.) Cell loss reaches a plateau after 12 hours of preconditioning, each seeded LVAD was subjected to an in vitro preconditioning regimen for 12 hours that exposed the cell lining to flow conditions(9). In brief, 12 hours before the in vivo experiments began, each LVAD was incorporated under sterile conditions into an in vitro flow loop (Figure 2). The in vitro flow loop was connected to a pneumatic drive console operated at 70 beats per minute and an ejection fraction of 30%. It has been demonstrated that cell loss reached a plateau after 12 hours of preconditioning a smooth muscle cell layer on LVAD surfaces (9). During the 12-hour preconditioning period, 18 samples of cell culture medium were collected (three each at 0, 0.3, 3, 5, 7 and 9 hours) to assess the amount of cell loss and to calculate preconditioning efficiency based on the number of initially seeded cells.

FIGURE 2 In vitro flow loop used for preconditioning of seeded cells. The loop consisted of a reservoir filled with cell culture medium (1) and the cell-seeded LVAD (2). Samples of cell culture medium emptied into the reservoir via a stop cock (3) were collected from the reservoir at several time points during the 12-hour preconditioning period as described in Materials and Methods. The in vitro flow loop was connected to a pneumatic drive console (not shown).

In Vivo ExperimentsAnimal Care
The tissue- donor calf used in the in vivo experiments received humane care in compliance with the Principles of Laboratory Animal Care prepared by the National Society for Medical Research and the Guide for the Care and Use of Laboratory Animals prepared by the Institute of Laboratory Animal Resources and published by the National Institutes of Health (NIH Publication No. 86-23, revised 1985).
LVAD Preparation
Immediately before implantation, one of the four seeded LVAD’s used in the in vitro experiments was disconnected from its preconditioning in vitro flow loop and discharged of cell culture medium. To eliminate any remaining cell culture medium, the LVAD was rinsed twice with pure RPMI medium and washed three times with 37ºC phosphate-buffered saline. Once the LVAD was filled with PBS, its inflow and outflow conduits were capped and its external surface sterilized by rinsing twice with 70% ethanol. The LVAD was then transported under sterile conditions to the operating room for immediate implantation.
LVAD ImplantationOperative Procedure.
Once prepared, the seeded LVAD was implanted into the tissue-donor calf under cardiopulmonary bypass using a standard procedure described previously (10). In brief, an abdominal incision was made through which the LVAD was placed. The LVAD’s percutaneous driveline was tunneled subcutaneously and exteriorized high on the left flank. The inflow and outflow conduits were passed through separate 1- to 2-cm-long incisions in the anterior left hemidiaphragm. A 20-mm-diameter low-porosity Dacron outlet graft was preclotted using autologous serum and blood and then anastomosed end-to-side at the descending thoracic aorta. The sewing ring of the LVAD was sutured to the left ventricular apex, using interrupted #2-0 braided polyester sutures with Teflon felt pledgets. A small crux incision was made in the apex, and a coring knife was inserted into the left ventricular cavity. A full-thickness circular segment of the apical myocardium was then excised. The pump inlet tube was inserted into the left ventricle. Air was removed from the LVAD via a needle inserted into the Dacron graft. Protamine sulfate was administered intravenously to antagonize heparin. The LVAD was kept in automatic mode at all times while implanted.
Postoperative Care.
Dextrose 5% in Ringer’s lactate solution was infused intravenously as necessary to maintain central venous pressure (CVP). Potassium chloride was added to the intravenous infusion as indicated by serial measurements of serum potassium. Sodium cefonicid 1 g was administered daily for 4 days to prevent infection. Butorphanol 10 mg was given i.m. every 4-8 hours for pain. No anticoagulative therapy was administered.
Necropsy and Gross Observations
Seven days after LVAD implantation, the calf was euthanized (by i.v. administration of 3 mg/kg heparin and then 1 ml/kg beuthanasia-D) and necropsied. The LVAD and pertinent organs (e.g., heart, lungs, liver, rumen, spleen, kidney, adrenal glands, and brain) were examined grossly and photographed. The organs were evaluated for the presence of emboli, ischemia, and infarction.
Scanning Electron Microscopy and Transmission Electron Microscopy
The LVAD was disassembled, inspected, photographed, and subjected to scanning electron microscopy (SEM) and transmission electron microscopy (TEM). In brief, both artificial surfaces of the LVAD’s, along with the attached cellular lining, were immediately fixed in Millonig’s phosphate buffer supplemented with 3% glutaraldehyde and incubated for several days at 4ºC. SEM samples were further fixed in 1% osmium tetroxide for 1 hour at room temperature. The samples were then rinsed with Millonig’s phosphate buffer and gradually dehydrated with ethanol (9).

For SEM, samples of the diaphragm and the titanium housing of the LVAD were coated with gold using a Denton Vacuum 502A Cold Sputter Module. The samples were then placed in a digital scanning electron microscope (Zeiss Model 960) to visualize the cell-lined sintered titanium and textured polyurethane surfaces.

For TEM, samples of the LVAD’s diaphragm and housing were infiltrated with resin and ethanol, embedded in the resin overnight, cut with a diamond knife to a thickness of 60-80 nm, pulse-stained in uranyl acetate and lead citrate, and finally viewed under a transmission electron microscope (JEOL Model 1200EX).