Minispheroids as a Tool for Ligament Tissue Engineering: Do the Self-Assembly Techniques and Spheroid Dimensions Influence the Cruciate Ligamentocyte Phenotype?

Ingrid Zahn,Clemens Gögele,Gundula Schulze-Tanzil,Tobias Braun

doi:10.3390/ijms222011011

Abstract

Spheroid culture might stabilize the ligamentocyte phenotype. Therefore, the phenotype of lapine cruciate ligamentocyte (L-CLs) minispheroids prepared either by hanging drop (HD) method or by using a novel spheroid plate (SP) and the option of methyl cellulose (MC) for tuning spheroid formation was tested. A total of 250 and 1000 L-CLs per spheroid were seeded as HDs or on an SP before performing cell viability assay, morphometry, gene expression (qRT-PCR) and protein immunolocalization after 7 (HD/SP) and 14 (SP) days. Stable and viable spheroids of both sizes could be produced with both methods, but more rapidly with SP. MC accelerated the formation of round spheroids (HD). Their circular areas decreased significantly during culturing. After 7 days, the diameters of HD-derived spheroids were significantly larger compared to those harvested from the SP, with a tendency of lower circularity suggesting an ellipsoid shape. Gene expression of decorin increased significantly after 7 days (HD, similar trend in SP), tenascin C tended to increase after 7 (HD/SP) and 14 days (SP), whereas collagen type 1 decreased (HD/SP) compared to the monolayer control. The cruciate ligament extracellular matrix components could be localized in all mini-spheroids, confirming their conserved expression profile and their suitability for ligament tissue engineering.

Highlights

Spheroids represent self-assembled three-dimensional (3D) cell aggregates with a round or ellipsoid shape
A total of 250 and 1000 L-CLs per spheroid were seeded as hanging drop (HD) or on an spheroid plate (SP) before performing cell viability assay, morphometry, gene expression and protein immunolocalization after 7 (HD/SP) and 14 (SP) days
Hoyer et al investigated the expression profile of lapine anterior cruciate ligamentocytes in large spheroids consisting of 3 × 105 cells, cultured under static and dynamic conditions in more detail and found distinct differences at the protein level induced by the respective culture conditions [14]

Summary

Introduction

Spheroids represent self-assembled three-dimensional (3D) cell aggregates with a round or ellipsoid shape. Spheroids can be produced using many cell types and techniques [1] including tenocytes and ligamentocytes [2,3,4] They can be used as 3D tumor models for the screening of agents and strategies effective to inhibit tumorigenesis [1,5]. Hoyer et al investigated the expression profile of lapine anterior cruciate ligamentocytes in large spheroids consisting of 3 × 105 cells, cultured under static and dynamic conditions in more detail and found distinct differences at the protein level induced by the respective culture conditions [14] This suggests that the culture type of spheroids but possibly the spheroid preparation technique and spheroid sizes might influence ligamentocytes expression profile. The maximum spheroid size is limited by the requirement of sufficient nutrient diffusion into the core of the spheroid (the nutrition limit is around 250 μm) [20]

Methods

Results

Discussion

Conclusion