Culture Techniques

Primary PDL cell cultures are established from explants of PDL tissues as previously described. Frequently, these teeth are obtained as teeth extracted from children and young adults undergoing orthodontic treatment. Following extraction, tissue for initiating a new culture is scraped from the middle one-third of a tooth using a sharp sterile blade. Usually, only a thin layer of tissue remains attached to the root following extraction, so the collected fragments usually require no additional dissection prior to explanting.

The harvested tissue explants are placed in culture medium consisting of Dulbecco's Modified Eagle's Medium (DMEM) containing 4,500 mg/L D-glucose, 584 mg/L L-glutamine, 110 mg/ml sodium pyruvate, 4 mg/L pyridoxine hydrochloride, 3700 mg/L sodium bicarbonate, and 10% bovine calf serum (hereinafter referred to as complete DMEM). This medium is used to both establish and maintain growth of PDL cells in culture. Most studies using these cells have reported supplementing the medium with FBS, however data from our laboratory have shown that it is unnecessary to maintain these fibroblast-like cells in FBS (Figure 3). The proliferation of PDL cells cultured using bovine calf serum appears identical to that of cells cultured using FBS. After collecting the explant, the PDL fragments are placed in a drop of complete DMEM with 1% penicillin/streptomycin and 1% fungizone in a 60mm tissue culture dish by tapping the blade gently against the floor of the dish. A stainless steel wire grid is placed on top of the fragments to prevent movement of the explant and facilitate outgrowth of the new cells onto the culture surface. Some studies have described using glass cover slips or directly placing the explant onto the plastic surface for a few minutes prior to adding medium as alternatives to promote explant adhesion to the surface [20]. In our laboratory, we have found a high rate of cell proliferation is achieved from explanted PDL tissues under wire grids (Figure 4). The explant and wire grid are gently covered with four ml of complete DMEM with antibiotics. The cultures are allowed to remain undisturbed for approximately 48 hours under 37°C and 5% CO 2 to permit attachment of explants. The complete DMEM with antibiotics remains in the cultures for the first three days. Medium is then replaced every 4-6 days until sufficient cell proliferation is evident. When feeding the culture (changing the medium), care is taken not to jostle the explant fragments. Generally, cells that have migrated out of tissue fragments and attached to the wire grid can be observed within one week using an inverted microscope. Depending on the size of the explanted tissue pieces, it takes 10-14 days for the maximum number of viable cells to migrate from the tissue. In approximately 10 days, about 2/3 of the cells are clustered on the wire grid surrounding the PDL tissue, and the migration rate starts to decrease. At this time the cells are subcultured. The cells are removed from the wire grid and culture dish by brief incubation (4-5 minutes) with 1 ml 0.25% trypsin, and are then transferred to a 100 mm tissue culture dish for continued growth.

The time to confluency in 100mm plates varies with the growth rate of the culture. In most preparations, confluency is usually reached after 1-2 weeks, at which time the cultures contain about 3-4 x 106 viable cells. Subculture from the 100mm dish is accomplished using a brief incubation with 2 ml of 0.25% trypsin. Once the cells have been successfully removed from the 100 mm dish, the cell number is halved by adding 1ml of cells directly from the trypsin suspension to a new culture dish containing 7ml of complete DMEM. The small amount of trypsin does not appear to affect the cells. Alternatively, the trypsinized cells can be counted using a hemacytometer or cell counter and reseeded at 0.5-1x106 cells per 100mm plate (72,500 cells/cm2).

Figure 3. Effects of different culture media on cell growth. Human PDL cells were seeded in 24-well plates (5,000 cells/well) in DMEM supplemented with 10% fetal bovine serum, 10% bovine calf serum, 10% iron-supplemented bovine calf serum, or 10% combined serum containing 85% bovine calf serum and 15% fetal bovine serum. Cell numbers (quadruplicate samples) were measured after two 48-hour periods, and every three days thereafter using a Coulter counter. Data are presented as mean ± standard deviation.

Figure 3. Effects of different culture media on cell growth. Human PDL cells were seeded in 24-well plates (5,000 cells/well) in DMEM supplemented with 10% fetal bovine serum, 10% bovine calf serum, 10% iron-supplemented bovine calf serum, or 10% combined serum containing 85% bovine calf serum and 15% fetal bovine serum. Cell numbers (quadruplicate samples) were measured after two 48-hour periods, and every three days thereafter using a Coulter counter. Data are presented as mean ± standard deviation.

Metal grid Glass slide Dry Floating in attachment medium

Initiating method

Figure 4. Comparison of four methods to initiate cell cultures. PDL tissue derived from each of four subjects was divided into four sections for culturing using one of four techniques. These techniques included the use of either a metal grid or glass slide to position the explant on the tissue culture surface, the dry attachment of the tissue to the plate surface by allowing the explant to sit on the plastic for 15 minutes prior to adding medium, or the explant left floating in the media. The data represent the percentage of explants that successfully produced cell outgrowth for each of the culturing techniques.

The population doubling time for successful PDL cell cultures at 37°C in DMEM containing 10% bovine calf serum is between 24 and 55 hours. Most primary PDL cell cultures grown in 10% bovine calf serum are capable of surviving 16-40 passages, or up to 150 days.

The primary PDL cultures do not retain their viability in continuous culture indefinitely. To preserve desired characteristics, cell stocks are frozen at early passage number. For the best recovery of frozen cells, subconfluent PDL cells are fed with fresh growth medium 24 hours before freezing. PDL cells are frozen at a concentration of 2-3 x 106 cells/ml in freezing medium (DMEM-supplemented medium containing 10% dimethyl sulfoxide and 10% bovine calf serum). Cells in freezing medium are dispensed into cryogenic vials and tightly sealed. The cells are slowly frozen by placing the cryogenic vials at 0°C for 2-3 hours, -20°C for 2-3 hours, and -80°C overnight, before transfer to liquid nitrogen for long-term storage. For successful revival of frozen cells, a vial is removed from the liquid nitrogen tank and thawed in a 37°C water bath as rapidly as possible. Then, the contents of the vial are transferred into a 100mm tissue culture plate containing 9 ml of growth medium. Cells are incubated at 37°C in humidified air containing 5% CO2 and the medium replaced after 24 hours.

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