Poster BB4 - Effects of donor and passage number on neocartilage derived from extensively passaged human costal chondrocytes

Introduction: As allogeneic cell-based therapies emerge, it is important to 1) select appropriate donors by characterizing cell sources for donor-to-donor variability and 2) determine an optimal or working passage number based on a balance of cell yield and functionality. In the U.S., the development of such human cell, tissue, and cellular and tissue-based products (HCT/Ps) regulated by the Food and Drug Administration (FDA) typically calls for the generation and characterization of cell banks for individual donors (i.e., no mixing of cells from different donors) and the investigation of a maximum working passaging number [1]. Functionally robust engineered neocartilage at passages as high as passage 7 (P7) has been fabricated from Yucatan minipig costal chondrocytes through the scaffold-free, self-assembling approach [2,3]. To translate such work to develop an HCT/P under the FDA regulatory framework, here it was hypothesized that processes optimized using minipig costal chondrocytes can be further optimized to fabricate robust engineered neocartilage from extensively passaged human costal chondrocytes. Furthermore, to investigate the effects of donor and passage number, it was hypothesized that the self-assembling process is robust against donor-to-donor variability and passages as high as P9. A two phased approach was used in this study. First, the protocol was optimized for human costal chondrocytes at low passage: P3. Second, functionality of human neocartilage fabricated from six individual donors was investigated at two passages: P5 and P9. This work is intended to guide the development of cell banks consisting of human costal chondrocytes for cartilage tissue engineering using allogeneic approaches.

Materials and

Methods: The objectives of this study were 1) to optimize the neocartilage fabrication process for human costal chondrocytes and 2) to investigate the effects of donor and passage on the functionality of neocartilage derived from human costal chondrocytes. Human costal chondrocytes were isolated from six donors (Table 1). Chondrocytes were conservatively passaged, cryopreserved at passage 1 (P1 master cell banks or P1 MCBs) and then further passaged and cryopreserved at P3 and at P7 as working cell banks (WCBs). Growth profiles of human costal chondrocytes were evaluated through population doublings (number cell doublings) and expansion factors. In Phase 1, engineered neocartilage (5 mm diameter) was fabricated at P3 from P1 MCBs for two donors through the processes of conservative passaging, aggregate rejuvenation (7 or 14 days), and self-assembly [2]. Engineered neocartilage was then evaluated morphologically and mechanically (creep indentation and tensile testing). In Phase 2, engineered neocartilage (5 mm diameter) was fabricated at P5 and P9 from P3 and P7 WCBs, respectively, using an optimized aggregate rejuvenation time from Phase 1 and the additional application of enzymes and growth factors [2]. Engineered neocartilage was evaluated morphologically (diameter, thickness, wet weight), histologically (H&E, SafO/FG, Picrosirius red), mechanically (creep indentation and tensile testing), and biochemically (total collagen and glycosaminoglycan [GAG]) [3]. Two-way analysis of variance tests (ANOVAs) were performed for factors of donor (six donors) and passage (P5 vs P9) with Tukey’s post-hoc test (p < 0.05) (GraphPad Prism). Statistically significant differences are indicated by the compact letter display format.

Results, Conclusions, and Discussions: Human costal chondrocytes from six donors were individually passaged up to P9, during which MCBs and WCBs were created for further evaluation. The high proliferative growth profiles of all six donors suggest that human costal chondrocytes are an effective cell source for allogeneic cell-based therapies (Table 1). Notably, donors A, B, and C, considerably younger in age than other donors, reached cumulative expansion factors in the tens of millions at P9. In Phase 1, compared to 14 days, 7 days of aggregate rejuvenation resulted in better mechanical properties as also previously reported for human articular chondrocytes [4], and thus 7 days of aggregate rejuvenation was carried to Phase 2. In Phase 2, WCBs were used to fabricate neocartilage from six donors individually at both P5 and P9. Morphological evaluation (Fig. 1) indicated neocartilage formation with flat morphologies and diameters greater than 5 mm except for donor F neocartilage. A fluid-filled sac formed for donor D at P5, while further passaged donor D P9 constructs appeared normal, indicating changes in cellular phenotype can happen across passages. Histological staining indicated intense staining for GAG and collagen (Fig. 2) with no overall differences for P5 vs P9. Mechanical properties (Fig. 3) also indicated that donor differences were significant, while passage number was not. For the compressive properties, aggregate modulus and shear modulus reached as high as 321 kPa and 138 kPa, respectively, and for tensile properties, Young’s modulus and UTS reached as high as 2.92 MPa and 1.19 MPa, respectively. Biochemical analysis of collagen and GAG (data not shown) indicated no differences among passages. These results indicate that 1) the self-assembling process is applicable to human costal chondrocytes at extensively high passages in producing robust engineered neocartilage and 2) master and working cell banks can be created from individual donors toward their use in allogeneic cell therapies.

Acknowledgements (Optional):