Using hyaluronan and proteoglycan link protein 1 (HAPLN1) and microfibrillar associated protein 5 (MFAP5) expression as indicator tool in classifying chondrocytes from fibroblasts

Inventors

Rapko, Stephen M. • Duguay, Stephen J.

Assignees

Vericel Corp

Abstract

Methods of evaluating the composition of a cell culture (e.g., to distinguish chondrocytes from fibroblasts) and methods for evaluating the phenotype of an individual cell (e.g., as a chondrocyte) are disclosed. The methods may be used, for example, for assessing chondrocyte cultures used for treatment of cartilage defects. In some embodiments, the invention involves identifying cell culture composition or the identity of a cell based on expression level of a fibroblast marker. In other embodiments, the invention involves comparing expression levels of at least one chondrocyte marker and at least one fibroblast marker in a cell culture sample or in an individual cell. In illustrative embodiments, the chondrocyte marker is hyaluronan and proteoglycan link protein 1 (HAPLN1), and the fibroblast marker is microfibrillar associated protein 5 (MFAP5).

Core Innovation

The invention relates to methods to evaluate the composition of a cell culture for chondrocytes and to determine a cell phenotype using expression levels of microfibrillar associated protein 5 (MFAP5) and, in some embodiments, one or more chondrocyte markers. A culture of cells for expansion of chondrocytes is obtained, a plurality of cells is selected, and the average expression level of MFAP5 is determined. The composition of the culture is then determined based on the average expression level of MFAP5, where an average expression level below a predetermined threshold indicates that the cell culture comprises chondrocytes.

In further embodiments, the invention determines the expression level of MFAP5 together with hyaluronan and proteoglycan link protein 1 (HAPLN1) or other chondrocyte markers, and determines the composition of the culture based on those expression levels. A cartilage biopsy is obtained from a mammal, cells are isolated, and the isolated cells are cultured in a cell culture to produce chondrocytes. In these embodiments, the expression levels of MFAP5 and HAPLN1 (or a selected chondrocyte marker) are determined in a plurality of cells, and the presence of chondrocytes is determined based on the expression levels.

The invention also covers evaluation approaches that confirm or identify the presence of chondrocytes or differentiate chondrocytes from fibroblasts and/or synoviocytes. For confirming presence, a plurality of cells is obtained from a cell culture derived from cartilage or synovium, and the average expression level of MFAP5 is compared to a predetermined threshold indicating that the culture comprises chondrocytes. For identifying fibroblasts and/or synoviocytes, the average expression level of MFAP5 above a predetermined threshold indicates that the culture comprises fibroblasts and/or synoviocytes.

In ratio-focused evaluation, the method performs a cell count and determines average MFAP5 expression to determine the ratio of chondrocytes to fibroblasts and/or synoviocytes, and optionally further determines expression of a chondrocyte marker to support the ratio determination. Additional embodiments determine ratios, including HAPLN1:MFAP5 ratios, and use predetermined threshold rules to indicate whether the culture contains more chondrocytes or more fibroblasts and/or synoviocytes.

Claims Coverage

The independent claims provide a core framework based on determining MFAP5 expression in a plurality of cells and using predetermined threshold rules to indicate whether a culture comprises chondrocytes, fibroblasts and/or synoviocytes, or to determine a ratio between these cell types. The claim set includes inventive features that add one or more chondrocyte markers, including HAPLN1, and define selectable marker sets and ratio-threshold relationships to refine composition evaluation.

Across the independent claims, the coverage centers on composition evaluation by measuring MFAP5 expression in a plurality of cells and comparing that expression to predetermined thresholds to indicate chondrocyte presence or fibroblast/synoviocyte presence. Additional independent-claim coverage includes evaluating MFAP5 together with HAPLN1 or another chondrocyte marker and determining the presence of chondrocytes, as well as determining the ratio of chondrocytes to fibroblasts and/or synoviocytes using MFAP5 threshold logic together with cell counting.

Stated Advantages

Documented Applications