Page 10 - IJB-8-3

Ultrathin Scaffolds for Monolayer RPE Cell Culture
RPE tissue engineering has been shown promise to cell culture and tissue engineering [17-20] . However, this
build RPE models for discovering therapeutics agents powerful technique has not been used to produce scaffold
and tissue transplant for patients with AMD [3-5] . In RPE for RPE regeneration because the current EHDJ-printed
tissue engineering, it is a great challenge to culture a scaffolds have large pore sizes (>50 μm), which are not
mature and functional RPE monolayer on scaffolds that suitable to hold the small RPE cells (tight size range
are biocompatible for transplant. At present, a commonly within 8–12 μm) . Due to Coulomb’s effect and stage
[21]
accepted approach for scaffold fabrication is to mimic the movement, it has been challenging to print scaffolds
[18]
human Bruch’s membrane, including the thickness and with smaller pore sizes (e.g., <50 μm) . This limits
fibrous structures that could promote cell attachment and the culturing of monolayer tissues, such as RPE on the
growth [3,6,7] . Bruch’s membrane is a selective permeable surface of the porous scaffold. In a few studies recently,
extracellular matrix that supports RPE and has thickness researchers have printed scaffolds with a pore size
of 2 – 4 μm in healthy human eyes . It is a great challenge <50 μm. For example, He et al. successfully fabricated
[5]
in mimicking the complex functionalities and structures scaffolds with a fiber diameter of 200 nm and pore size
of Bruch’s membrane in vitro. of 10 μm . This printing method was assisted with
[22]
Tremendous efforts have been put into developing indium tin oxide glass, which made the scaffold hard to
new RPE culture systems from the perspectives of be removed from the glass. Nevertheless, those results
designing and fabricating culturing devices [4,8-10] . One of demonstrate a great promise of obtaining scaffolds that
the most important approaches is film casting, which can satisfy the requirements of RPE regeneration by EHDJ
quickly produce porous membranes with thickness close printing. Herein, we reported our findings on developing
to Bruch’s membrane [2,5,10] . At present, this technique the EHDJ printing method for ultrathin scaffolds
is widely used to build the commercialized permeable with small pore sizes by optimizing EHDJ printing
inserts, including Transwell and Millicell cell culture process parameters and applying the scaffolds for RPE
®
®
insert [11-13] . Permeable membranes of cell culture inserts monolayer culture.
were successfully transplanted into a non-human
primate model . However, those membranes share 2. Materials and methods
[14]
several drawbacks, including sheet-like morphology and 2.1. Materials
undesirable mechanical properties. Another common
approach to fabricate scaffolds for RPE is electrospinning, Polycaprolactone (PCL) pellets with a molecular weight
which can produce membranes with fibrous nature similar of 50,000 kDa were purchased from Perstorp Inc.
to that of Bruch’s membrane with suitable thickness [3,6] . (Capa™ 6500, Sweden). Glacial acetic acid (AcOH) in
However, due to randomly oriented and highly packed HPLC grade was purchased from Macklin Inc. (A801303,
structures, the capability of diffusion is greatly limited ≥99.9%, China). PCL ink with a 60% w/v concentration
when the thickness of electrospun scaffolds is greater than was prepared by mixing PCL pellets in AcOH and
2 μm . Conventional fabrication technologies for RPE dissolving the mixture at 60°C for 3 h through a 100 W
[10]
cell culture can produce ultrathin structures. However, the ultrasonication treatment. The solution was stored in an
internal order-less microstructures of those scaffolds have incubator for degassing at 26°C for 2 h. Subsequently, the
random pore distribution and poor interconnection and not ink was transferred to a syringe equipped with a stainless
possible to reproduce scaffolds. Three-dimensional (3D) needle before printing. All the stainless nozzles used are
printing technology can overcome this problem because 24-gauge with an approximate outer diameter of 0.57 mm
it ensures the structure of the printed membranes highly and an inner diameter of 0.31 mm. Polyester-terephthalate
®
orderly and repetitive at micrometer precision. Such high (PET) Transwell with 0.4 μm pore membrane inserts
precision and reproducibility are critical not only for were purchased from Corning Inc. (3470, USA).
research purpose but also essential for commercial scale 2.2. EHDJ printing system
production of scaffolds.
Electrohydrodynamic jet (EHDJ) printing is a An EHDJ printing system developed in-house was used
mature technology that features high precision and to conduct small pore size scaffold fabrication . The
[16]
well-defined structure . It can produce well-orientated EHDJ printing system (Figure 1A) was built with an
[15]
micro-/nano-scale fibrous scaffolds with precise structure XYZ motion stage (Aerotech Inc., USA), a high-voltage
and shape control. EHDJ utilizes an electric field to pull power supply (DC voltage from 0 to 10 kV, Dongwen Inc.,
fine fibers from the printing nozzle and control the fiber China), a single-channel syringe pump (NE-1000, New
orientation with high-precision moving stage . The Era Pump System Inc., USA), and a digital microscope
[16]
scaffold structure can be designed by changing the stage (B011, Supereyes, China). A silicon wafer (Ferrotec,
moving path or optimizing key process parameters. The Japan) was used as a scaffold collector attached to the stage
printed scaffolds have been successfully applied in 3D fixed on the X-Y motion plane. The ambient parameters

2 International Journal of Bioprinting (2022)–Volume 8, Issue 3