Clinical Specimens: Studies were approved by the Institutional Review Board. Control specimens were obtained from the Cooperative Human Tissue Network and surgical specimens from patients undergoing surgical disc procedures. Cells used in the study of monolayer vs 3D ECM matrix production assessed with immunohistochemistry were derived from the lumbar annuli of a 67 year old female donor and from four patients (two 35 year old males, a 53 year old male, and a 76 year old female). Cells used in the study of the mitogenic responsiveness to PDGF in monolayer or 3D culture were derived from 12 subjects, mean age 47 years (± 13.9 yrs (S.D.)); 7 were males, 5 were females. The disc sites were lumbar in 9 subjects and cervical in 3. Cells grown on Matrigel™ were derived from two cervical and two lumbar discs from two donors and two surgical patients (mean age 43 years; 2 males and 2 females). For all subjects studied, the mean Thompson score for gross disc morphology was grade III .
Cell Culture Methods: Primary cultures were grown as previously described [11
12]. Following primary culture, cells were subsequently passaged in either monolayer culture (seeded at 5,000 cells/well of a Lab-TekR Chamber SlideTM (Nunc, Napierville, IL) or into 3D culture in 1.2% Keltone LV alginate (Kelco, San Diego, CA) at a cell density of 50,000 cells seeded into the alginate which was then layered onto a Costar Transwell Clear Insert (Costar, Cambridge, MA). Cells were grown as previously described in sterile modified Minimal Essential Medium with Earle's salts, non-essential amino acids, penicillin-streptomycin and 20% fetal bovine serum . Passages 1-4 were studied in parallel for ECM produced after 10 days of culture.
Analysis of ECM Production with immunohistochemistry: After 10 days of culture, alginate inserts were fixed in 1% neutral buffered formalin for 5 minutes, dehydrated, embedded in paraffin and serially sectioned en face. Monolayer cultures were fixed with cold acetone for 1 minute and stored in the cold until immunolocalization was performed.
Immunohistochemical localizations utilized rabbit anti-human collagen Type I (1:100 dilution) or rabbit anti-human collagen Type II (1:150 dilution) (Biodesign International, Kennebunk, ME), monoclonal anti-proteoglycan delta DI-4S (ICN, Costa Mesa, CA), and monoclonal anti-keratan sulfate (Seikagaku Corp., Tokyo, Japan) using previously described techniques . Negative controls were processed minus primary antibodies and also minus secondary antibodies. Localization utilized DAB colorimetric visualization of ECM components.
Analysis of ECM localization products in 3D cultures was performed using computer-assisted quantitative histomorphometry with the OsteoMeasure software (OsteoMetrics, Inc., Atlanta, GA). An average of 240 colonies/passage/subject were evaluated. Data are presented as mean ± s.e.m.
Analysis of Mitogenic Response to PDGF: Cells were fed 0.5 ml per well minimum essential medium with Earle's salts prepared as described above except that fetal bovine serum was supplemented at 1% (MEM1) to constitute the control culture conditions (1% serum is necessary for healthy cell maintenance. Platelet-derived growth factor (PDGF; Sigma-Aldrich, St. Louis, MO) was added to MEM1 to achieve a final concentration of 100 ng/ml PDGF, a dose shown in prior work to have anti-apoptotic effects on disc cells . For monolayer culture, cells were seed at a density of 16,000 cell/well in a 48 well culture plate; for agarose 3D culture, cells were seeded at a density of 50,000 cells/well in a 24 well culture plate and prepared as previously described. Cells grew for 10 days. DNA synthesis was measured by pulse labeling cultures with 2 μCi/ml of [3H]-thymidine (Amersham) during the last 24 hours of culture; aliquots were taken and analyzed to determine total DNA content using a fluorometric procedure as previously described [11, 16]. Results were expressed as cpm [3H]-thymidine uptake/μg DNA. Each cell cultures was assessed in 3-5 replicate samples and results averaged.
Culture on Matrigel™: Cells from four subjects were derived as described above, trypsinized and plated at a density of 5,000 cells/well onto Nunc 8-well slides coated with 75-100 μl Matrigel™ (Becton Dickinson, Bedford, MA) diluted 1:1 with MEM with 20% FBS. Control wells were seeded with the same density of cells on uncoated wells. Cells were allowed to grow for 8 days, fixed with 1% neutral buffered formalin, rinsed with 70% ethanol, stained with toluidine blue, coverslipped and evaluated with light microscopy.
Statistical analysis: Statistical analysis of the 3D immunohistochemistry ECM data utilized SAS software (version 6.11, SAS Institute Inc., Cary, NC). A repeated measures analysis of variance was performed to test for differences over time; all tests were two-sided and p-values < 0.05 were considered statistically significant. Data are expressed as mean ± s.e.m.; paired t-tests were used in analyses of cell proliferation..