Three-dimensional culture systems to induce chondrogenesis of adipose-derived stem cells

Abstract

Stem cells can easily be harvested from adipose tissue in large numbers for use in tissue-engineering approaches for cartilage repair or regeneration. In this chapter, we describe in vitro tissue-engineering models that we have used in our laboratory for the chondrogenic induction of adipose-derived stem cells (ASC). In addition to the proper growth factor environment, chondrogenesis requires cells to be maintained in a rounded morphology in three-dimensional (3D) culture, and thus properties of the biomaterial scaffold also play a critical role in ASC differentiation. Histologic and immunohistologic methods for assessing chondrogenesis are also presented. In general, 10-12 weeks are required to assess ASC chondrogenesis in these model systems.

Figure 1

(a) appearance of ASC pellet 24 hours after centrifugation in the bottom of a 15 ml conical tube, (b) Left: chondrogenic and Right: control pellets after 8 weeks of in vitro culture. Note: magnification differ between (a) and (b).

Figure 2

(a) ASC pellet in alginate before mixing, (b) micro stir bar introduced to alginate, (c) micro stir bar prior to mixing, and (d) micro stir bar during mixing. Note that no bubbles are introduced in 2 ml of solution using this method. Note: as low as ~ 700 μl can be used with this method without introducing bubbles.

Figure 3

(a) Preparation of pipette tip for making alginate beads - size of bead can be controlled depending on where the tip is cut, (b) Dropping alginate beads into 102 mM CaCl₂, and (c) cross-linked alginate beads in a 24 well plate.

Figure 4

Set up for agarose/ASC construct preparation. Magnetic stirrer with hot plate is configured with temperature feedback control: agarose temperature control solution (black arrow) and sterile agarose to be used for experiment (white arrow).

Figure 5

Preparation of agarose cultures, (a) dispensing 2% agarose using a 14 gauge needle onto ASC pellet, (b) Injecting molten agarose into custom mold, and (c) agarose/ASC gel after 10 minute gelation at room temperature.

Figure 6

(a) punching 6 mm diameter discs with a skin biopsy punch, and (b–c) 6 mm diameter ASC-seeded agarose discs after punching.

Figure 7

ASC-seeded agarose discs (6 mm diameter) in a 24 well plate in 1 ml of medium/well.