The Scar-in-a-Jar has been developed in our laboratory in order to address the problems of in vitro
fibrogenesis discussed above. This system: (1) solves the problem of tardy collagen deposition; (2) quantitatively measures relative changes in collagen I deposition via immunofluorescence and in situ
optical analysis; (3) allows for high content screening within a single well including α-SMA and another ECM protein of choice; and (4) while implementing normalization for cell number by counting nuclei via 4',6-diamidino-2-phenylindoldilactate (DAPI) staining [59
]. As discussed above, the addition of charged and neutral macromolecules into culture medium dramatically enhances the deposition of collagen into the pericellular matrix [61
] (Figure ), within a shorter time, to a greater extent and degree of cross-linking than possible in both fibroplasia models by Clark et al
. (1997) [63
] and Fish et al
. (2007) [47
] (Figure ). In both cases, enhanced collagen deposition reduces culture time to 48 h (DxS) and 6 days (Fc), prior to optical quantitation. TGF-β1 addition is optional. For optical analysis, samples in a 24-well format are immunostained for collagen I and fibronectin, and cell nuclei are stained with DAPI. Automated image acquisition using a 2× objective is performed and the area of collagen I and fibronectin per cell is ascertained using the Metamorph®
Imaging System software (Figure ).
The Scar-in-a-Jar enables the analysis of proteins-of-interest and cell enumeration all within a single-well, thus minimizing processing steps, material loss and sample variation. Cell enumeration can give an indication of potential anti-proliferative effects of a compound and allows for correction of protein production to account for the variance in cell numbers due to that compound. We also demonstrated its capability to discern the reduction of deposited collagen by known and novel inhibitors targeting various points of the collagen biosynthesis pathway from the epigenetic to the extracellular level, including two C-proteinase inhibitors, FSY002, originally developed in a German pharmaceutical company [89
] and PCP56 from Pfizer (NY, USA) [47
]. PCP56 demonstrated efficacy in agreement with an earlier report on the abridged fibroplasia model [47
] but FSY002 did not work in our system. FSY002 has an interesting history. This phosphinate inhibitor did inhibit purified C-proteinase in the test tube and an IC50
value could be obtained [89
]. However, tests in conventional monolayer fibroblast culture proved inconclusive and we have to conclude in hindsight that this was due to tardy C-proteinase activity. Running this substance in our system with fully active C-proteinase activity revealed its ineffectiveness. At this point in time, optical analysis is unable to discern a reduction in collagen cross-links if there is no net collagen reduction on the cell layer [59
]. Hence, the testing of lysyl oxidase inhibitors will be better analysed by the biochemical analysis method employing pepsin digestion of cell layers followed by SDS-PAGE and silver staining to visualize collagen cross-links (Figure ) [85
The combination of short culture time, rapid collagen biosynthesis, complete deposition, together with optical analysis that circumvents the need for protein extraction, makes the Scar-in-a-Jar a convenient assay that remains in a single well from start to finish.
Besides the current use of fibroblasts, this system has the flexibility and potential to screen the effect of potential antifibrotic compounds on other organ specific culprits such as hepatic stellate cells. The optical accessibility of the Scar-in-a-Jar has room for the introduction of one or more additional cell types like monocytes or smooth muscle cells to augment the fibrotic context and tailor it for a dermal wound healing situation or an atherosclerotic plaque. This could be done in direct coculture or using inserts, with the fibrotic target cells adhering to the bottom of the well. Besides preliminary testing of exogenously added matrix metalloproteinases [59
], the current system has not yet been fathomed for its ability to study collagen matrix metalloproteinases turnover that would be relevant for analysing effects of collagen cross-link inhibitors and inducers of matrix metalloprtoteinases like hepatocyte growth factor. We envision longer observation times in this instance or challenging cell cultures after an initial ECM build-up, with additional cell types or substances that induce remodelling. A very promising line of research using this system could be non-enzymatic glycation studies to capture a diabetic situation and testing breakers of advanced glycation end products.