Culture and Cryopreservation of Chondrocytes from Human Cartilage: Relevance for Cartilage Allografting in Otolaryngology

Abstract

One of the reasons for failure of cartilage allografts is the impaired condition of the transplant during storage. In this paper we describe methods for the isolation and culture of viable chondrocytes obtained from nasal septum cartilage. Furthermore, we evaluate the possibility of growing such specific chondrocytes under culture conditions and storing them in a frozen state. Age dependent differences were observed in the growth rate of the cultured cells. Our results confirm that chondrocytes survive freezing and remain able to proliferate. Knowledge gained from this study may be applied to the culture and freezing of viable intact cartilage for use in reconstructive surgery in otolaryngology.

Human Immunodeficiency Virus Cannot Productively Infect Freshly Cultured Human Cartilage Cells

“…The cartilage specimens were processed as previously described [8], Briefly, cartilage was freed of perichondrium and finely minced. The extracellular matrix w'as digested for 12-18 h at 37 °C in the presence of 2 mg/ml type II collagcnasc (Seromed, Berlin, FRG), 0.1 mg/ml hyaluronidasc (Serva, Berlin), and 0.15 mg/ml DNase (Paesel, Frankfurt, FRG) in RPMI 1640 medium (Seromed), in small spinner flasks with agitation until intact fragments were no longer visible.…”

“…The cells were grown in complete medium, which consisted of RPMI 1640 containing 20% fetal calf serum (FCS) as described elsewhere [8], To characterize the cell type present in the cultures, immunocytochemical staining was performed.…”

Synthesis of Human Cartilage Using Organotypic Cell Culture

“…Small numbers of cells can be cultured and expanded by passage using conventional enzymatic techniques. This also has the ad vantage that cells may be cryopreserved in liquid nitrogen for future growth [10]. The rapid modulation of the colla gen phenotype that occurs following the first subculture, that is to say the rapid decline of type II collagen synthesis and initiation of type I and type III collagen synthesis [13.…”

“…The cartilage specimens were processed as previously described [ 10]. Briefly, cartilage was freed of perichondrium and finely minced.…”

Basic Fibroblast Growth Factor and Insulinlike Growth Factor I Support the Growth of Human Septal Chondrocytes in a Serum-Free Environment

“…The remaining samples were then minced into 1-to 3-mm 3 cubes, placed into a spinner flask, and incubated at 37°C in a digestion medium for 18 to 36 hours (Figure 1, B). A modification of a previously described digestion medium 12 was used and consisted of type II collagenase (2.00 mg/mL), hyaluronidase (0.10 mg/mL), and type I deoxyribonuclease (0.15 mg/mL) (all from Worthington Biochem Corp, Freehold, NJ) in Dulbecco modified Eagle medium (DMEM) mixed 1:1 with Ham F12 medium (Gibco, Grand Island, NY). After digestion, the dispersed cells were filtered through a 40-µm nylon cell strainer (Becton Dickinson, Franklin Lakes, NJ) to remove any remaining undigested clumps.…”