Agar overlay technique vs. v-bottom plate: differentiation of human cartilage-like microtissues depend on plate format

Introduction:

Since the self-repair capacity of joint cartilage defects is limited, several cell-based therapies were developed for the treatment by using e.g. chondrocytes isolated from patient biopsies. However, chondrocytes lose their characteristic properties during 2D culture in vitro. A 3D environment supports the redifferentiation of these cells and the production of a hyaline-like extracellular matrix, though with varying success depending on e.g. the donor and the used method for microtissue generation. The aim of this study was to compare the chondrogenic maturation of chondrocyte-derived microtissues using two distinct approaches.

Methods:

Human chondrocytes isolated from knee joints were expanded in medium with human serum. Scaffold-free microtissues were generated using two different approaches: the agar overlay technique and pellets induced in v-bottom plates. After two and four weeks the macroscopic appearance (reflected-light microscopy) was documented and the size was determined. The differentiation degree was evaluated via histology to visualize typical glycosaminoglycans (Safranin O, Alcian blue) and immunohistochemistry (IH) to detect cartilage-specific markers (collagen type II, proteoglycans, Sox9) and collagen type I on cryosections. The metabolic activity of microtissues was determined via the quantification of the ATP level.

Results:

Cells from all donors in both experimental set ups generated microtissues – but with distinct differences in the differentiation levels. Microtissues cultured in v-bottom plates showed a higher differentiation level compared to microtissues on agar. This is evidenced by a higher amount of matrix resulting in bigger microtissues (diameter on v-bottom plates is 80% larger compared to agar) and increased metabolic activity. Cartilage-like composition of the matrix (histology) showed little/local colouring on agar versus intense/overall staining on v-bottom plates. IH analysis of cartilage-specific markers showed the same pattern. The expression level of collagen type II and proteoglycans was enhanced in microtissues from v-bottom plates. However, Sox9 expression is distributed ubiquitously in both cultivation approaches. The dedifferentiation marker collagen type I was similarly expressed on a low level. Absolute values vary from donor to donor.

Discussion:

In our study, the redifferentiation of chondrocytes in microtissues is directly linked to the method for the induction and development of cell aggregates. The chondrogenic profile of microtissues in v-bottom plates is superior to that on plates covered with agarose. Therefore, v-bottom plates are an alternative for the generation of microtissues without an influence of a foreign substance. These microtissues seem to be quite suitable as transplants to regenerate cartilage defects.