Challenges of aortic valve tissue culture - maintenance of viability and extracellular matrix in the pulsatile dynamic microphysiological system

Abstract

Background: Calcific aortic valve disease (CAVD) causes an increasing health burden in the 21^st century due to aging population. The complex pathophysiology remains to be understood to develop novel prevention and treatment strategies. Microphysiological systems (MPSs), also known as organ-on-chip or lab-on-a-chip systems, proved promising in bridging in vitro and in vivo approaches by applying integer AV tissue and modelling biomechanical microenvironment. This study introduces a novel MPS comprising different micropumps in conjunction with a tissue-incubation-chamber (TIC) for long-term porcine and human AV incubation (pAV, hAV).

Results: Tissue cultures in two different MPS setups were compared and validated by a bimodal viability analysis and extracellular matrix transformation assessment. The MPS-TIC conjunction proved applicable for incubation periods of 14-26 days. An increased metabolic rate was detected for pulsatile dynamic MPS culture compared to static condition indicated by increased LDH intensity. ECM changes such as an increase of collagen fibre content in line with tissue contraction and mass reduction, also observed in early CAVD, were detected in MPS-TIC culture, as well as an increase of collagen fibre content. Glycosaminoglycans remained stable, no significant alterations of α-SMA or CD31 epitopes and no accumulation of calciumhydroxyapatite were observed after 14 days of incubation.

Conclusions: The presented ex vivo MPS allows long-term AV tissue incubation and will be adopted for future investigation of CAVD pathophysiology, also implementing human tissues. The bimodal viability assessment and ECM analyses approve reliability of ex vivo CAVD investigation and comparability of parallel tissue segments with different treatment strategies regarding the AV (patho)physiology.

Keywords: Calcific aortic valve disease; ECM remodelling; Microphysiological system; Tissue culture; Viability.

Fig. 1

Three-layered aortic valve tissue in histology and schema: HE, Alcian blue and Movat Pentachrom stain illustrate dominating ECM components in fibrosa, spongiosa and ventricularis layer of aortic valve leaflet covered with endothelial cells. Valvular interstitial cells are responsible for ECM maintenance in non-diseased tissue (Scale bar: 100 µm)

Fig. 2

Experimental workflow TIC-MPS and control AV tissue culture: pAVs or hAVs (not shown) are excised, cut into 3 × 5 mm.² specimens and deployed to dynamic setup using a high flow (upper chip layout) or low flow (lower chip setup), static or lysis control that are incubated in conventional 24-well culture plates (Scale bar: 5 mm)

Fig. 3

MPS-TIC Setup: A The MPS control unit enables regulation of pumping sequence and pneumatic pressure; B The high flow setup is conjoined with the attenuation element (here shown without culture medium); C The AV tissue specimen is stitched to a TPU ring and inserted into the tissue incubation chamber (TIC) (Arrows: flow direction [Q], scale bar: 1 cm). D Example of a stitched (TPU ring) pAV specimen prior insertion in the TIC

Fig. 4

Shear force calculation: A Navier Stokes derivation is used to compute the shear forces inside a circular tube that act on the AV-tissue specimen (τ≙wall shear stress, μ≙dynamic viscosity, d≙diameter, Q≙ flow rate)

Fig. 5

Pulsatile dynamic culture medium propulsion: The pneumatic pump cycle is illustrated and the flow rates in high flow setup (A) and low flow setup (B) were measured by particle image velocimetry (PIV). Cycle-specific shear forces were computed (filled circle: pneumatic pressure applied, empty circle: vacuum applied; Abscissa: seconds)

Fig. 6

Resazurin reduction viability assessment in dynamic and static AV tissue culture: A pAV tissue specimens were incubated for 14 days. Non-invasive resazurin reduction viability assessment was performed on days 0, 4, 8, 10, 12 and 14. (n = 6); B hAV tissue sections were incubated for 26 days. Resazurin reduction assays were conducted every second day. (n = 3); C AV from dynamic setup stitched to the TPU ring after 2 h of static viability assessment (two-way ANOVA; * p < 0.05, vertical asterisks for respective setup)

Fig. 7

Visualization of native pAV tissue resazurin penetration: The dashed indicator bar displays peak penetration depth with high resorufin concentration. A The fluorescing resazurin reduction product resorufin is visible in red. B The DAPI-stained nuclei impose blue-fluorescence (n = 3, representative samples shown, Scale bar: 200 µm)

Fig. 8

End-point viability assessment by LDH cryosection stain after pulsatile dynamic vs. static AV tissue culture: A LDH-viability stain performed on pAV tissues at the beginning of the experiment and following 14 days of incubation under high flow dynamic, low flow dynamic, static and death tissue conditions. (Left to right, representative samples shown, n = 6, scale bar: 500 µm); DAPI nuclear counterstain is visualized in the lower part of the tissue section; B Positively stained areas were quantified and relativized against cross section surface; C Staining intensity variation was metrically assessed by applying a colour threshold (two-way-ANOVA; * p < 0.05; # p < 0.1)

Fig. 9

Mass reduction and tissue shrinkage in AV tissue culture settings: A pAV tissue mass was measured at the beginning of the experiment and after 14 days of incubation; B Cross section areas before and after the experiment (14 days) were opposed (n = 6; two-way-ANOVA; * p < 0.05; no significance shown for lysis control)

Fig. 10

Cellular density after pulsatile dynamic vs. static AV tissue culture: A Density alteration is visible in DAPI stained nuclei of native (0 days, left) and statically incubated pAV tissue Sects. (14 days, right). B Cellular density was assessed and relativized against AV cross section area due to valvular shrinkage (n = 6, Scale bar: 300 µm; two-way-ANOVA; * p < 0.05; no significance shown for lysis control)

Fig. 11

Collagen quantification and tissue laminae after pulsatile dynamic vs. static AV tissue culture: A pAV tissues were stained with picrosirius red at the beginning of the experiment and after 14 days of high and low flow dynamic, static and lysis incubation conditions to reveal collagen fibres (left to right, representative samples shown, n = 3, scale bar: 500 µm); B MOVAT pentachrome stain was applied to assess trilaminar tissue stratification (C). A sponge-like ECM configuration appeared in lysis control (D); E Collagen fibres were quantified according to picrosirius red stain and relativized against cross section surface (two-way-ANOVA; * p < 0.05)

Fig. 12

α-SMA and CD31 immunohistochemistry staining of dynamic vs. statically cultured AV tissue sections: A Statically incubated pAV specimen after 14 days is shown (left, n = 3, representative sample shown); B Preliminary AV incubation setups under static conditions demonstrated increased focal α-SMA expression after 21 days; C CD31 epitopes of pAV tissue specimens are stained after 14 days of static incubation. The magnification shows neo-endothelial lining; D Quantification of CD31 expression (n = 3, scale bar: 200 µm; two-way-ANOVA; * p < 0.05)