33 Embryonic Limb Mesenchyme Micromass Culture as an In Vitro Model for Chondrogenesis and Cartilage Maturation Anthony M, DeLise, Emanuela Stringa, Wendy A. Woodward, Maria Alice Mello, and Rocky S. Tuan 1. Introduction In vitro techniques for the study of chondrogenic differentiation of embryonic limb mesenchymal cells have been available for some time. Early methods require high- density confluent monolayer cell cultures (1,2). The micromass culture method devel- oped by Ahrens et al, (3) represented a convenient system for the observations and analysis of the differentiative processes and phenomena analogous to those exhibited by the limb cartilage anlagen in situ. In these cultures, limb mesenchymal cells first undergo condensation giving rise to aggregates that later become cartilage nodules (3,4), thereby mimicking the differentiative phenomena occurring during embryonic limb development in vivo, i.e., mesenchymal condensation preceding cartilage differ- entiation (5-9). The micromass limb mesenchymal culture system has gained great popularity for the analysis of the regulatory steps and differentiative processes that result in the condensation of the mesenchyme and the formation and maturation of the cartilage anlagen. This chapter outlines the protocols developed for culturing limb bud mesenchymal cells from mouse and chick embryos used in the authors’ laboratory. In addition, tech- niques and protocols for histochemical and immunohistochemical detection of chon- drocyte markers at all stages of differentiation and maturation are also described. 2. Materials and Methods 2.1. Mouse Embryonic Limb Mesenchyme Cultures 2.1.1. Materials 1, Mouse embryos: Use gestational day 11.5 to 12.5 embryos (see Note 3.1.1.) 2. 10X Glucose: Composition per 250 mL, 2.5 g tissue culture grade glucose q.s. to 250 mL. with ddH,O, filter sterilize, and store at 4°C. 3. 10X Calcium-magnesium-free saline (CMES): Composition per 250 mL. 0.925 g KCI, 0.075 g KH,PO,, 20.0 g NaC, 0.568 g NaHCOj, 0.315 g NaHsPO, + H;0 qs. to 250 mL. with d4H,0, filter sterilize, and store at 4°C.. 200X Penicillin/streptomycin: Dissolve 2.89 g of pei iin (1730 Ulmg, Sigma Chemical Co., St. Louis, MO) and 5.0 g of streptomycin (761 U/mg, Sigma) in 500 mL. of water, pH to 7.3 and filter sterilize. Aliquot and store at ~20°C. . 1X Saline-glucose solution (CMFSG); Composition per 250 mL, 25 mL of 10X glucose, 25 mL of 10X CMFS, 1.25 mL of 200X penicillin/streptomycin, 4.5. to 250 mL with 44GH,O, filter sterilize, and store at 4°C. . Culture medium: Composition per 250 mL. 25 mL fetal bovine serum (Atlanta Biologicals, Atlanta, GA), 1.25 mL 200X penicillin/streptomycin, q.s.to 250 mL with 1X Dulbecco's Modified Eagle's Medium (DMEM; Gibco-BRL, Gaithersburg, MD). Store the prepared culture medium at 4°C. Digestion solution: 0.002% EDTA (Fisher Scientific, Pittsburgh, PA) and 0.01% trypsin type II from porcine pancreas (Sigma) in CMFSG. 20 jim mesh Nitex filter (Tetko, Lancaster, NY). ). Microdissection forceps and scissors. ). Stereomicroscope. . Hemacytometer. .2. Methods . Harvesting of the embryos: Sacrifice the mouse by cervical dislocation, remove the un. 12, B. | Change medium every other da embryos from the uterus by Caesarean section. Rinse embryos in CMFSG st room temperature. . With the aid of a stereomicroscope, dissect the limbs (both fore- and hindlimbs) using microdissection forceps and scissors. Collect the limbs in a $0-mL. conical tube with 4 mL CMFSG. . Cell dissociation: Add the digestion solution and pipet up and down using a 10-mL pipet for about 20-30 times, until the limbs are totally disintegrated (see Note 3.1.2.) . Stop the enzymatic digestion with an equal volume of culture medium. ass the mixture of dissociated limbs through the Nitex filter to eliminate fibrous materi- als and tissue debris. .. Take a 10-pL aliquot and determine cell number by hemacytometer counting and collect Resuspend the cell pellet in enough culture medium to reach the cell concentration desired {Grom 10 to 20 x 10° cells/mL). ). Plate cells as 10 pL micromass drop in a 24-mm tissue culture well and incubate at 5% CO;, 37°C in a humidified tissue culture incubator for 1.5 or 2h wo allow cell attuch- ment (see Notes 3.133, 3.1.4,, and 3.1.5.) Feed the micromass with 1 mL. of culture medium (see Note 3.1.6). Change medium after 24 h. After another 24 h, change medium with medium containing 25 g/mL ascorbic acid (see Note 3.1.7,2.1.2. Methods 10. M. 12, 13, 14 Harvesting of the embryos: Sacrifice the mouse by cervical dislocation, remove the embryos from the uterus by Caesarean section Rinse embryos in CMFSG at room temperature. With the aid of a stereomicroscope, dissect the limbs (both fore- and hindlimbs) using microdissection forceps and scissors. Collect the limbs in a 50-mL conical tube with 4 mL CMFSG. Cell dissociation: Add the digestion solution and pipet up and down using a 10-mL pipet for about 20-30 times, until the limbs are totally disintegrated (see Note 3.1.2.) ‘Stop the enzymatic digestion with an equal volume of culture medium, Pass the mixture of dissociated limbs through the Nitex filter to eliminate fibrous materi- als and tissue debris, Take a 10-pL aliquot and determine cell number by hemacytometer counting and collect the rest of the cells by centrifugation at 230g for 10 min. Resuspend the cell pellet in enough culture medium to reach the cell concentration desired (Grom 10 t0 20 x 108 cells/mL). Plate cells as 10 lL micromass drop in a 24-mm tissue culture well and incubate at 5% CO, 37°C in a humidified tissue culture incubator for 1.5 or 2 h to allow cell attach- ment (see Notes 3.1.3., 3.1.4,, and 3.1.5.) Feed the micromass with I mL. of culture medium (see Note 3.1.6.). ‘Change medium after 24h Afier another 24 h, change medium with medium cont (see Note 3.1.7,). Change medium every other day. ing 25 jigimL. ascorbic a