Xeno-Free Human Wharton's Jelly Mesenchymal Stromal Cells Maintain Their Characteristic Properties after Long-Term Cryopreservation

Abstract

Objective: The past decade has witnessed a rapid growth in harnessing the potential of adult stem cells for regenerative medicine. An investigational new drug (IND) or a regenerative medicine advanced therapy (RMAT) product must fulfil many requirements, such as stability studies, after cryopreservation. Such studies are important to ascertain the utility of off-the-shelf allogeneic cells for clinical applications. The present work describes a complete characterisation of xenofree human Wharton's Jelly mesenchymal stromal cells (hWJ-MSCs) before and up to 28 months post-cryopreservation.

Materials and methods: In this experimental study, culture methods that involved plasma derived human serum and recombinant trypsin were used to develop clinical grade cells. Complete cell characterisation involved flow cytometry studies for viability, positive and negative markers, colony forming unit (CFU) potential, population doubling time (PDT), soft agar assay to evaluate in vitro tumourigenicity, karyotype analysis and differentiation studies which were performed before and at 6, 12, 18 and 28 months post-cryopreservation.

Results: Our data showed consistency in the flow cytometry, CFU assay, PDT, soft agar assay, karyotyping and differentiation studies.

Conclusion: Using our protocols for extended xeno-free culture and cryopreservation of hWJ-MSCs, we could establish the shelf life of the cell-based product for up to 28 months.

Keywords: Mesenchymal Stromal Cells; Stability; Umbilical Cord; Wharton’s Jelly.

Fig.1

Human Wharton’s jelly mesenchymal stromal cells (hWJ-MSCs) migrating after 7-10 days of explant culture as observed under an inverted microscope (phase contrast: X4). The adherent cells appear typically fibroblastic. The first cells to migrate out of the tissue are denoted as passage-0 cells.

Fig.2

Flow cytometry studies for cell viability, and positive and negative markers. A. Mean cell viabilities and B. Haematopoietic and non-haematopoietic markers. The difference between the lots, passages and time points was not significant (data for five different lots; two-way ANOVA, P>0.05).

Fig.3

Representative images for flow cytometry studies across early and late passages, and at different time points. A. Cell viability remained unchanged, and B. Positive markers CD105 and CD90 were >95% in keeping with International Society for Cellular Therapy (ISCT) definitions for the mesenchymal stromal cells (MSCs).

Fig.4

Colony forming unit (CFU) assay and population doubling time (PDT) were evaluated. A. The mean CFU potential at early and late passages, and at different time points (data for three different lots; two-way ANOVA, P>0.05). The stemness of the cells was retained despite cryopreservation. B. Depicts the mean PDT across five lots, at early and late passages and different time points, which remained largely unchanged except for the anticipated lot to lot variability.

Fig.5

The soft agar assay was carried out to evaluate in vitro tumourigenicity. A. In vitro soft agar assay showed that after long-term culture and cryopreservation, the Wharton’s jelly mesenchymal stromal cells (WJ-MSCs) did not exhibit altered growth characteristics of plastic adherence and could not grow in soft agar. However, the positive control, the MCF7 breast cancer cell line, showed vigorous growth in soft agar and was not anchorage dependent, and B. Representative karyotype (46, XX) of WJ-MSCs. Long-term culture and cryopreservation did not affect the karyotype.

Fig.6

Representative images of differentiation studies of Wharton’s jelly mesenchymal stromal cells (WJ-MSCs). A. Osteogenic, B. Chondrogenic, and C. Adipogenic lineages.