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Ultrathin Scaffolds for Monolayer RPE Cell Culture
Bruch’s membrane structure plays an essential role in were quantified on the S20 scaffold (Figure 7E). Studies
the selectivity of nutrients and wastes in the outer blood- have shown that Na /K -ATPase is significantly reduced
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retinal barrier, thus it is important to mimic the actual in aged mice and that TEP plays a functional role in cell
permeability of the native membrane. The difference migration, division, polarization, and development .
[33]
of cells and the stage of maturity of the monolayer of These data are consistent with a conclusion that S20
RPE may account for the large P . Nonetheless, it is scaffold may have the ability to provide a better growing
app
remarkable that all the culturing substrates had decreased environment for the RPE cells that manifest higher
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permeability over time after RPE cell seeding, suggesting expression of Na /K -ATPase and increased TEP.
a steady gain of barrier function conferred by RPE cells
on the scaffolds. Particularly, probe molecules pass freely 4. Conclusions
through the pores of blank S50 scaffolds. After culturing, The first EHDJ-printed Bruch’s membrane mimic
the monolayer cell formation is likely responsible for the was designed and made for integration into the
drop of permeability. current commercial freestanding structures. The PCL
The TEER level is another important indicator for the scaffolds showed similar biomimetic properties to
barrier function of cultured RPE monolayer (Figure 7C). Bruch’s membrane, including native-like thicknesses,
[30]
Before culturing, all substrates were measured with a biomechanical properties, and permeability. It was
culture medium for calculating background resistance determined that the S20 PCL scaffold had better
(Table 2). PET membrane has the most negligible performances in culturing a monolayer of ARPE-19 cells.
porosity, which leads to the highest background TEER Moreover, PCL scaffolds have good biodegradability and
reading of 73.7 Ω·cm . The TEERs of S20 and S50 were can mimic extracellular matrix environments in vivo;
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around 63.1 Ω·cm and 50.2 Ω·cm , respectively. The therefore, they are conducive to RPE maturation. Taken
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resistances measured of S20 and PET membrane were together, our results demonstrated that the EHDJ printing
comparable throughout the entire duration and reached technique can fabricate scaffolds mimicking Bruch’s
a plateau on day 14 with a TEER at around 47 Ω·cm . membrane with high resolution and precision, and the
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Similar readings have been reported using immortalized printed scaffolds have great potential to help RPE cells
RPE cells grown on Transwell membrane and electrospun form mature tissue with the desired functionality. Future
scaffolds [11,31] . For RPE cultured on S50, the TEER reached work can explore the design and fabrication of scaffolds
the same level as the PET membrane and S20 at the end suitable for culturing embryonic stem cell-derived RPE
of the third culture week. This phenomenon suggested for transplantation.
that cell sinking or collapsing in the pore could affect its
formation of barrier function as proper monolayer was not Funding
developed. Nevertheless, it also could reach the same level This work was financially supported by Key Program
of TEER after 3 weeks of culturing. In the human body, Special Fund in Xi’an JiaoTong-Liverpool University
RPE together with retinal vascular endothelium forms (XJTLU) under Grant KSF-E-37. This work was also
the blood-retinal barrier, which is closely associated supported by the National University of Singapore
with photoreceptor outer segments. Thus, EHDJ-printed (Suzhou) Research Institute under an internal grant to the
scaffolds are promising for in vitro culture of RPE to build Center for Peak of Excellence on Biological Science and
tight junctions and finally present barrier function. Food Engineering.
The barrier function and selectivity of RPE benefited
from the cooperation of different proteins and their Conflict of interest
physiological functions, including Na /K -ATPase and
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tubulins. Na /K -ATPase can regulate sodium gradient The authors have no conflicts of interest to declare.
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formation for osmotic gradient and water diffusion [32,33] . Author contributions
Then, the tight junction complex proteins (such as ZO-1)
at the boundaries of neighboring RPE cells will increase Conceptualization, J.S., D.H., and H.L.; data curation:
the resistance between the apical and the basal sides of the H.L., F.W., K.Y.; investigation: H.L., F.W., R.C., and
cells. Therefore, a high concentration of Na will usually Y.C.; methodology: H.L., L.J., J.S., and D.H.; software:
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accumulate at the apical side of RPE and form a TEP H.L. and K.Y.; validation: H.L., F.W., R.C., Y.C., and
difference. In our case, the TEP difference in S20 scaffold T.L.; writing – original draft: H.L.; writing – review
is significantly higher than that grown on PET membrane and editing: D.H., X.S., B.V.S., Z.L, J.S., F.W., Y.C.,
and S50 scaffolds (Figure 7D). Thus, the expression of R.C., K.Y., L.J, T.L., and H.L; funding acquisition:
Na /K -ATPase in RPE cells cultured on S20 should be D.H. and J.S.; project administration: D.H. and J.S.;
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higher than RPE cultured on PET membrane and S50. and supervision: D.H. and J.S. All authors reviewed and
As predicted, increased protein levels of Na /K -ATPase commented on the manuscript.
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12 International Journal of Bioprinting (2022)–Volume 8, Issue 3