Speaker
Description
Introduction:
Bioreactors are an important tool to study clinical scenarios under more realistic conditions before heading towards preclinical and clinical studies. We previously showed that, using our standard bioreactor (SB), multiaxial load activates transforming growth factor β1 (TGF-β1), and thereby pushes the mesenchymal stromal cells (MSCs) towards chondrogenesis[1]. Such bioreactors offer the unique opportunity to test tissue engineered constructs under kinematic loading conditions. However, only 4 samples can be run at the same time. Therefore, we developed a multi-well bioreactor (MSB) which allows for the simultaneous multi-axial loading of 16 samples at the same time with additional advantages, such as ease of handling. In this study, we compare the MSB to the SB and showcase how it could be used for further studies revolving around higher throughput testing of cell-laden constructs used in the field of tissue engineering.
Methodology:
MSCs were obtained from three donors after acquiring written consent from the patients (Freiburg, EK-326/08) and encapsulated in fibrin–poly(ester-urethane) scaffolds (5 x 106 cells per scaffold). Samples were cultured in TGF-β1-free chondropermissive medium either in an unloaded state (UL) or subjected to joint-mimicking multiaxial load (combination of shear and compression) within two different bioreactors: a previously used standard bioreactor (SB) and a new multi-sample bioreactor (MSB). The loading protocol consisted of 1 h of loading for 20 days during a period of 25 days. Culture medium was collected and replaced every second to third day. ELISAs were performed to quantify active and total produced TGF-β1. Nitrite was detected using the Griess assay and sulphated glycosaminoglycans (sGAG) were quantified using DMMB assay. The different outputs were all normalized to the DNA content (measured with the Hoechst dye) of the respective samples. Additionally, for time points at day 7 and day 25, gene expression was quantified using real time PCR (both standard plates and array cards) and histological sections were stained using Safranin O / Fast Green (SAFO-FG).
Results:
TGF-β1 was activated only within the SB and MSB groups. MSB showed greater sGAG retention within the samples loaded compared to both SB and UL samples (p < .001). In contrast, for MSB samples, the total sGAG content (medium + sample) was lower than in SB and UL samples (p < .001). Gene expression revealed an increase in chondrogenic markers (ACAN, COL2, SOX9/RUNX2 ratio) for both SB and MSB compared to UL. MSB samples also showed more enhanced SAFO-FG staining compared to both SB and UL.
Discussion:
We highlight the use of a novel multi-well bioreactor that performs similarly if not better than a standard bioreactor in terms of mechano-induced chondrogenesis. Furthermore, this multi-well bioreactor offers the unique opportunity to apply kinematic like loading to multiple samples at the same time in a user-friendly manner, which could lead to great advantages for swift testing of TE constructs.
[1] Li, Z., et al. Tissue Eng Part A 15, 1729-1737 (2009).
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