Primary culture of Stem Cells from Apical Papilla (hSCAPs) of the human premolar and third molar teeth
Briefly, human dental papilla tissue was procured from discarded 6 ~ 24 aged donor’s premolar and third molar teeth with informed consent of patients undergoing routine extractions at the Dental Clinic of School of Dentistry in Kyung Hee University, under approved guidelines set by the Kyung Hee University and School of Dentisty Human Subjects Research Committees (IRB# KHUSD 0908–01). The extracted teeth were directly stored in alpha-MEM (Lonza) containing 1% penicillin/streptomycin (P/S, Lonza), and the teeth were used to procure papilla tissue within 2 hours after teeth extraction. Dental apical papilla tissues were extracted from the premolar and third molar teeth, and were minced with a scalpel. The fragmented papilla tissues were washed with phosphate buffered saline (PBS: Gibco) three times, and the minced tissues were allowed to attach on T25 tissue culture flasks in basic culture medium consisting of alpha-MEM, 10% fetal bovine serum (FBS, lonza), and 1% penicillin/streptomycin. Cultures were fed every 2 days and passaged by treatment with 0.25% trypsin/EDTA. Cell Cultures were grown at 37°C in a humidified atmosphere containing 5% CO2.
Morphological and immunocytochemical characterization of hSCAPs
1 × 104 cell/cm2 of hSCAPs were seeded on 24 well plate (Corning) and cultured for 2 days. The attached cells were fixed with 3.7% formaldehyde (Sigma) for 20 mins at room temperature, and fixed cells were washed with PBS. The cells were permeabilized with 0.2% Triton X-100 (Sigma) treatment for 20 mins, and, after brief washing with PBS, were treated with 4% bovine serum albumin (BSA: Sigma) to block non-specific binding of antibodies at 4°C for overnight. After blocking, the cells were incubated with primary antibodies such as 1:200-diluted mouse-anti-nestin (Abcam) antibody, 1:200-diluted mouse-anti-Stro-1 (Abcam) antibody, and 1:200-diluted mouse-anti-CD44 (Abcam), and 1:200-diluted mouse-anti-CD133 (Abcam) at 4°C for overnight. After reaction with primary antibodies, the cells were washed gently with PBS three times, and then incubated with secondary antibodies such as 1:2000-diluted alexa 488 (goat anti-mouse IgG, Invitrogen), 1:2000-diluted alexa 596 (goat anti-mouse IgG, Invitrogen) for 1 hr at room temperature under dark condition. Finally, after brief washing with PBS three times, the cells were counter-stained with DAPI, and mounted. The stained cells were observed under an inverted fluorescence microscope (Olympus, IX-72).
Detection of mycoplasma contamination
Mycoplasma contamination of primary cultured hSCAPs was evaluated by mycoplasma specific staining method and polymerase chain reaction (PCR) method.
For mycoplasma staining, cell suspension was prepared by trypsin-EDTA treatment for 5 minutes and subsequent neutralization with culture media. Mycoplama staining was processed with prepared hSCAPs suspension and Mycoplasma Detection Kit according to manufacturer’s instruction. Briefly explaining, 95 μl of cell suspension (1 × 105 cell/ml) was mixed with 5 μl of MycoFluor™ Mycoplasma Detection Kit (Invitrogen) in micro-centrifuge tube, and the mixed cell suspension was incubated for 30 mins at 37°C in a 5% CO2 incubator. After incubation, the stained sample was observed under an inverted fluorescence microscope (Olympus, IX-72).
To evaluate mycoplasma contamination in gene level, PCR was processed with BioMycoX Mycoplasma PCR Detection Kit (Cells-safe) according to manufacturer’s instruction. Briefly, after 24 hrs of culture, 1 ml of culture media was collected, and centrifuged at 3000 rpm for 5 mins. After centrifuge, supernatant was transferred to microcentrifuge tube, and was centrifuged at 13,000 rpm for 10 mins. After centrifuge, mycoplasma pellet was collected, and suspended with 100 μl of dH2O, and then boiled at 98°C for 10 mins. 50 μl of supernatant was transferred into new PCR tubes. For PCR reaction, supernatant was mixed with 5 μl PCR template, 2 μl primer mix (Cells-safe), 2 μl dH2O and 10 μl 2X PCR premix (Cells-safe). PCR reactions were performed as following conditions: pre-denaturation process at 95°C for 5 mins with 1 cycle, denaturation process at 94°C for 30 secs with 35 cycles, annealing process at 55°C for 30 secs with 35 cycles, and extension process at 72°C for 30 secs with 35 cycles. Finally, all PCR products were loaded on 0.7% argarose gel containing ethidium bromide at concentration of 0.5 μg/ml.
Mycoplasma elimination
Mycoplasma elimination of contaminated hSCAPs was processed using BioMycoX® Mycoplasma Elimination Kit (Cells-safe). After detection of mycoplasma contamination, 5 × 106 of the mycoplasma-infected hSCAPs was seeded on a T75 flask and cultured in culture media at 37°C in a 5% CO2 incubator. When the confluence of cells reached at 70 ~ 80%, the cells were trypsinized, neutralized with culture media, and then centrifuged at 1500 rpm for 5 mins. After centrifuged, cell pellet was collected, and then suspend with culture media containing 5% FBS. 2.5 × 106 cells/ml was mixed with 200 μl of BioMycoX® Reagent 1, and total volume was adjusted to 10 ml with culture media. The cell suspension was transferred to a T75 flask, and incubated for 3 days without media change at 37°C in a 5% CO2 incubator. After 3 days of incubation, attached cells were trypsinized, and then centrifuged at 1500 rpm for 5 mins. After centrifuge, cell pellet was collected and suspended with culture media containing 10% FBS. Cell suspension was mixed with 200 μl of BioMycoX® Reagent 2, and total volume was adjusted to 10 ml with culture media. The cells were incubated for 2 days without media change at 37°C in a 5% CO2 incubator, which treatment with BioMycoX® Reagent 2 was repeated two times.
Proliferation assay of mycoplasma-infected and -eliminated hSCAPs
Mycoplasma-infected hSCAPs and mycoplasma-eliminated hSCAPs were seeded at cell density of 1 × 104 cells on 96 well plate and incubated for 24 hrs at 37°C in a 5% CO2 incubator. The BrdU incorporation based cell proliferation assay was processed with BrdU assay kit (Merck) according to manufacturer’s instruction. Briefly, after 24 hrs of incubation, 1:2000 diluted BrdU working solution was added into hSCAPs culture media, and the cells were incubated for 3 hrs. The BrdU incorporated cells were treated with 200 μl of fixative/denaturing solution for 30 mins at room temperature. After reaction, fixative/denaturing solution was aspirated, and the cells were incubated with 1:100 diluted mouse anti-BrdU in dilution buffer solution for 1 hr at room temperature. After washing with washing buffer three times, the cells were reacted with 100 μl goat anti-mouse IgG HRO conjugate for 30 mins at room temperature, and then washed with washing buffer three times. Finally, the cells were reacted with 100 μl of substrate solution in the dark at room temperature for 15 mins, and subsequently 100 μl of stop solution was added to each well. The content of incoporated BrdU in cellular DNA was measured using a spectrophotometric plate reader at dual wavelengths of 450–540 nm (or 450–595).
2D and 3D osteogenic differentiation of mycoplasma-eliminated hSCAPs
Prior to osteogenic differentiation, primary cultured hSCAPs were expanded in α-MEM supplemented with 15% FBS, and 1% penicillin/streptomycin. For osteogenic differentiation, hSCAPs was seeded on culture plated at cell density of 1 × 105/cm2 and cultured in osteogenic medium; α-MEM supplemented with 15% FBS, and 1% penicillin/streptomycin containing 50 μg/ml ascorbic acid (Sigma) in PBS, 1 μM dexamethasone (Sigma) in PBS and 10 mM β-glycerophosphate (Sigma) in PBS. The cultures were maintained at 37°C in a 5% CO2 humidified incubator and fed every 2 days up to 20 days. After 20 days of osteogenic culture, the differentiated cells were fixed with 3.7% formaldehyde for 1 min or 20 mins at room temperature, and fixed cells were washed with PBS. The fixed cells were stained with phenotypic alkaline phosphatase (ALP) staining kit (Chemicon) and alizarin red-S (Sigma) staining solution according to manufacturer’s instruction.
In addition, for 3D osteogenic differentiation, hSCAPs were suspended in 1.1% (w/v) alginic acid (Sigma) and 0.1% (v/v) porcine gelatin (Sigma) solution (all dissolved in PBS, pH 7.4), as described before [19]. Briefly, the cell-gel solution was passed through a peristaltic pump (EYELA) and dropped using a 25-gauge needle into sterile alginate gelation solution composing of 100 mM CaCl2 (Sigma), 10 mM HEPES (Sigma), and 0.01% (v/v) Tween (Sigma) at pH 7.4 with stirring. Approximately 10,000 cells were encapsulated in each alginate hydrogel. The hydrogels remained in gently stirred CaCl2 solution for 6–10 mins and were then washed with PBS. The hSCAPs containing hydrogels were transferred to in 10 ml vessels of HARV bioreactors (Synthecon) and the vessels was rotated at 25 rpm. Subsequently, 3D osteogenic differentiation was induced using the same osteogenic medium described above. The bioprocess is illustrated in Figure 2 II. After 3D osteogenic differentiation, the sections of hydrogels were stained with alizarin red-S staining solution according to manufacturer’s instruction.
2D and 3D neural differentiation of mycoplasma-eliminated hSCAPs
Prior to neural differentiation, primary cultured hSCAPs were expanded in culture medium consisting of DMEM/F12 mixture (1:1, with glucose, L-glutamine, HEPES Buffer, Lonza), 10% FBS, 20 ng/ml epithelial growth factor (EGF, Peprotech), 10 ng/ml basic fibroblast growth factor (FGF2, Peprotech), 1000 unit/ml LIF (Invitrogen) and 1% penicillin/streptomycin. For neural differentiation, hSCAPs was allowed to form neurospheres by plating non-adherent culture dishes in the presence of 100 ng/ml FGF2 in culture medium consisting of DMEM/F12 mixture, 10% FBS, 1000 unit/ml LIF (Invitrogen) and 1% penicillin/streptomycin for 3 days at 37°C in a 5% CO2 humidified incubator. The formed neurospheres were replated on laminin (10 μg/ml, Invitrogen)-coated culture dishes and were allowed to direct neural differentiation in neurobasal medium (Gibco) supplemented with 2% B27 (Invitrogen), 1X insulin-transferrin-sodium-selenite (ITS, BD science), 1% penicillin/streptomycin, 30 ng/ml of NGF, 20 ng/ml EGF and 10 ng/ml FGF2 for one week. Subsequently, hSCAPs were maintained in neuroinduction medium consisting of in neurobasal media (Gibco), 1% B27 supplement (Invitrogen), 1% non-essential amino acid (Gibco), 1 X ITS, 100 units/ml penicillin and 100 μg/ml streptomycin, 100 ng/ml GNDF (Peprotech) and 0.5 μM retinoic acid (Sigma). All of the media were replaced twice a week.
In addition, for 3D neural differentiation, hSCAPs which dissociated from neurospheres were mixed with matrigel (BD Bioscience), and the cell/matrigel solution and 1.1% (w/v) alginic acid solution were placed in separated syringes on syringe pump, and the solutions passed through double jet nodule and dropped into sterile alginate gelation solution composing of 100 mM CaCl2, 10 mM HEPES, and 0.01% (v/v) Tween at pH 7.4 with mild stirring. The hydrogels remained in gently stirred CaCl2 solution for 6–10 mins and were then washed with PBS. The bioprocess is illustrated in Figure 2 IIIE. The hSCAPs/matrigel were placed in inner side and alginate hydrogel were formed to be outer layer enveloping cell/matrigel. The 3D neural differentiation of hSCAPs containing hydrogels was induced by the culture under same culture condition described before. After 2D and 3D neural differentiation, the cells were fixed with 3.7% formaldehyde for 20 mins at room temperature, and neural differentiation was characterized by immunocytochemical staining with primary antibodies such as Ca2+/calmodulin-dependent protein kinases II (rabbit anti-human Cam kinase II, Abcam) and βIII-tubulin (mouse anti-human βIII-tubulin, Abcam).