Multiscale reverse engineering of the human ocular surface


Here we present a miniaturized analog of a blinking human eye to reverse engineer the complexity of the interface between the ocular system and the external environment. Our model comprises human cells and provides unique capabilities to replicate multiscale structural organization, biological phenotypes and dynamically regulated environmental homeostasis of the human ocular surface. Using this biomimetic system, we discovered new biological effects of blink-induced mechanical forces. Furthermore, we developed a specialized in vitro model of evaporative dry-eye disease for high-content drug screening. This work advances our ability to emulate how human physiological systems interface with the external world, and may contribute to the future development of novel screening platforms for biopharmaceutical and environmental applications.

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Fig. 1: A reverse engineered human ocular surface.
Fig. 2: Tissue engineering of the human cornea and conjunctiva in the biomimetic eye model.
Fig. 3: Recapitulation of eye blinking and physiological biomechanical environment of the ocular surface.
Fig. 4: An engineered model of evaporative dry eye.
Fig. 5: Evaluation of therapeutic efficacy of lubricin.

Data availability

The data sets that support the findings of this study are available from the corresponding author upon reasonable request. All requests for raw and analyzed data and materials are promptly reviewed by the University of Pennsylvania to verify whether the request is subject to any intellectual property or confidentiality obligations. Any data and materials that can be shared will be released via a Material Transfer Agreement.


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We thank G. Al, R. Dana and P. Argüeso for their input to this study; I. Gipson at the Schepens Eye Research Institute for providing immortalized human conjunctival epithelial cells; G. Jay at Brown University for providing human lubricin; K. Kwon for providing data on the kinematics of blinking in the human eye; D. Song, J. Vance, D. Caggiano, M. Henderson and J. DuPont for assistance in OCT, TearLab and keratography; L. Du and M. Allen for assistance in mechanical testing; and P. McClanahan for assistance in fluorescence imaging of tear fluids. This work was supported by the National Institutes of Health (NIH) grant nos. 1DP2HL127720-01 (to D.H.), R01EY026972 (to V.Y.B.) and K08EY025742-01 (to V.L.), the National Science Foundation grant no. CMMI:15-48571 (to V.B.S. and D.H.), the Research to Prevent Blindness (to V.Y.B.) and the University of Pennsylvania. D.H. is a recipient of the NIH Director’s New Innovator Award and the Cancer Research Institute Technology Impact Award.

Author information




J.S. designed the research, performed the experiments and analyzed the data with assistance from W.Y.B., A.G. and Y.-S.Y., and wrote the manuscript. In collaboration with J.S. and D.H., F.A. and V.B.S. developed and analyzed a theoretical model of the engineered eye model. M.M.-G., V.L. and V.Y.B. conducted clinical studies of patients with DED and helped J.S. and D.H. collect and analyze tear osmolarity, keratography and fluorescein staining data. D.H. designed the research, analyzed the data and wrote the manuscript.

Corresponding author

Correspondence to Dongeun Huh.

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Competing interests

D.H. holds equity in Emulate Inc. and consults for the company.

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Peer review information: Brett Benedetti was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 Formation of corneal and conjunctival epithelia using 3D cell patterning technique.

The concentric pattern of the ocular surface epithelia was replicated by plating human corneal and conjunctival epithelial cells on the surface of the scaffold following the formation of keratocyte-laden stroma. a, A 3D cell patterning technique is enabled by precisely controlled spreading of a cell suspension solution on the dome-shaped scaffold depending on the distance between a concave well and the convex surface of the scaffold. b, At a large distance (D), a cell-suspension solution sandwiched between the concave well and the convex scaffold forms a liquid bridge that wets the center of the scaffold and its vicinity. The meniscus of the solution is marked with a dotted line in the figure. c, When the well is brought in closer proximity to the scaffold (d « D), the liquid bridge spreads outward in the radial direction to increase the wetting area. d, A cell suspension containing corneal cells (green) is dispensed at the bottom of the concave well, which is subsequently inverted and positioned over the dome scaffold to bring the solution in contact with the convex surface of the scaffold. Once contact is established, the device assembly is kept in a humidified cell culture incubator to allow the seeded corneal epithelial cells to adhere to the surface of the scaffold. e, After cell attachment, the same procedure is performed using a solution containing conjunctival epithelial cells (red). In this process, the distance between the concave well and the scaffold is reduced to spread a suspension over the entire scaffold surface and to deposit the conjunctival cells on the peripheral region of the scaffold.

Extended Data Fig. 2 Responses of Transwell dry-eye model to desiccating stress.

The capacity of conventional in vitro platforms to model dry eye was investigated using air-liquid interface (ALI) culture of primary human corneal epithelial cells and keratocytes in Transwell inserts. a, This in vitro model was constructed as a Transwell equivalent of the eye model by creating a thin layer of collagen hydrogel interspersed with keratocytes on the porous membrane of the insert and then plating corneal epithelial cells on the surface of the hydrogel layer. Before induction of dry eye, the tissue construct was cultured submerged for 3 d and then maintained at the ALI for another 10 d to induce differentiation and stratification of the epithelium. bd, Simulation of evaporative dry eye in the Transwell model. b, (Control) The tissue was maintained in a regular humidified cell culture incubator (37 °C air, 100% RH). c, (Condition 1) The tissue constructs were moved to the DED simulation chamber to expose them under the same condition used for modeling dry eye in the eye model (25 °C air, 32 °C for culture medium and 50% RH). d, (Condition 2) Desiccating culture conditions previously reported in Transwell-based in vitro models of evaporative dry eye (40 °C air, 30% RH) were used. eg, Evaluation of the response of the Transwell dry-eye models to the desiccating environment using fluorescein staining after 4 d exposure. In the Control group (e), no fluorescence was detected in the central regions of the epithelium when treated with fluorescein. Similarly, the ocular surface tissues produced in Conditions 1 (f) and 2 (g) showed the absence of fluorescein staining despite their exposure to the desiccating environment. RH, relative humidity.

Supplementary information

Supplementary Information

Supplementary Methods, Supplementary Table and Supplementary video 1–3 legends

Reporting Summary

Supplementary Video 1

Supplementary Video 1 shows the key components of the engineered human ocular surface model and demonstrates how eye blinking and tear film formation are simulated in the device.

Supplementary Video 2

Supplementary Video 2 shows a top-down view of a hydrogel eyelid (blue) sliding over the engineered ocular surface. The eyelid is actuated at 0.2 Hz to match the frequency of physiological spontaneous blinking in the human eye. The movie is played in real time.

Supplementary Video 3

Supplementary Video 3 shows the digitally controlled DED simulation platform depicted in the Supplementary methods. The entire system is set up in a temperature-controlled cell culture incubator that contains a computer-controlled humidity sensor and humidifier to regulate the relative humidity of air in the surrounding environment of the DED model. The engineered device is mounted vertically on a custom-designed stage equipped with a heating pad and temperature probe. Two programmable syringe pumps are connected to the access ports of the device to perfuse the culture chamber with media and to inject artificial tears into the tear channel. The movie is played in real time.

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Seo, J., Byun, W.Y., Alisafaei, F. et al. Multiscale reverse engineering of the human ocular surface. Nat Med 25, 1310–1318 (2019).

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