An Indian Bioprinting Startup is Working on 3D Printed ‘Liquid Cornea’ for Corneal Grafts

In the last few years, there has been a continuous growth of bioprinting companies around the world, probably because the medical field is one of the most exciting industries taking advantage of 3D printing. After all, health is one of the top priorities for most people and when some of the top biotech startups create soft human tissue-like structures, vascular networks and three-dimensional liver tissue constructs for experimentation, curiosity strikes. Along with some of these technical milestones in 3D printing is Bengaluru-based firm Pandorum Technologies’ successfully engineered cornea tissue. Thanks to their cell-laden hydrogel, they can promote scarless healing of corneal wounds by regeneration. Like most firms in the industry, Pandorum is pushing the technology hoping to eventually become part of the no donor required utopia. We must caution our readers however that from a thing becoming possible in bioprinting and 3D printing for medicine it may take several decades before it is available to you and me. 

Pandorum’s bio-engineered cornea

The cornea is a thin piece of transparent tissue on the front of the eye that resembles a soft contact lens and is vulnerable to disease and injury. The World Health Organization (WHO) estimates that corneal opacities accounted for 7% of the world’s blind population in 2010, making it the third most common cause of blindness. And over 12 million people are waiting for a corneal transplant globally while 20% of childhood blindness is estimated to be caused by the cornea. In India alone, bilateral corneal blindness affects 1.2 million people and there are not enough donated corneas for transplants. A few years back a survey globally quantified the considerable shortage of corneal graft tissue, with only one cornea available for 70 needed, and although efforts are still strong for cornea donation everywhere, it is also crucial for bioengineering to look for alternative solutions.

Pandorum’s novel hydrogel uses a combination of specialized stem cells and bio-mimetic that can be directly applied in a minimally invasive manner as ‘Liquid Cornea’ to corneal wounds and perforations and used to print transparent and suturable corneal lenticules embedded with live corneal cells for therapeutic applications and future human implantation to treat visual impairment. The bioengineered extracellular matrix with corneal cells developed as ‘Liquid Cornea’ and Corneal Graft for Human implantation is a regenerative approach towards vision restoration through tissue engineering. It is designed for a simple, minimally-invasive application procedure, to reduce the need for post-operative medication and care.

The tissue engineering and regenerative medicine startup company announced the development of their bioengineered cornea tissue study at the annual meeting of the Association for Research in Vision and Ophthalmology (ARVO) held in Vancouver, Canada, last April. Where Tuhin Bhowmick, co-founder of Pandorum said that “being able to bioengineer critical tissues such as the human cornea is a significant milestone.” And while the work is currently in the animal studies stage, the research team is getting ready for their first pilot human trials in 2020. Founded in 1928, ARVO is the largest eye and vision research organization in the world that includes nearly 12,000 researchers and clinicians from over 75 countries.

“Though surgically replacing the opaque tissue with a clear corneal allograft is usually effective in improving vision, there is an acute shortage of cadaveric human corneas available for transplantation. In India alone, there are over a million people suffering from a bilateral loss of vision due to corneal disorders, and at least a few folds more from unilateral corneal blindness. At Pandorum, we are working to close this gap using a bioengineering approach through stage-wise development of a platform, which is ultimately aimed to liberate us from the dependencies on human donor cornea,” suggested Bhowmick.

It’s quite a big deal for an Indian company to do such groundbreaking work since the Asia Pacific is the fastest growing 3D bioprinting region in the global market due to the presence of a huge patient population and continuously developing economies, including India and China, with growing healthcare industries. According to the Indian Brand Equity Foundation, in 2017 the Indian healthcare sector was one of the fastest growing industries, advancing at an annual growth rate of over 22% since 2015 and is expected to reach 130 billion dollars in 2022.

But Pandorum is not entirely new to bioprinting, it was the first Indian company to 3D print a liver tissue and their work is focused on tissue engineering and regenerative medicine, a much broader field, with the final goal to develop both liver and cornea as lab-grown transplantable tissues. The company can generate tissues that comprise 10,000 to 1 million cells, still it could take up to five years to build the working prototype of a tissue that has billions of cells and can be transplanted into a patient -which will need to have its own blood vessel structure and be able to integrate within the receiver’s body. “Right now, our mini-liver tissue reflects the characteristics of those grown within the human body. So pharma companies can test the hepatotoxicity of drugs, or FMCG companies their products, nixing the need for animal trials,” explained Pandorum co-founder Arun Chandru last year in an interview with Forbes India.

Pandorum scientists working at the lab on tissue engineering

The startup incubated at the Bangalore Bioinnovation Centre (BBC), located at the National Centre for Biological Sciences campus is making tremendous progress in tissue engineering. Since it’s foundation in 2011 by academic entrepreneurs from the Indian Institute of Science, they have focused on research and development of artificial human organs, and have become quite a household name in India’s new bioprinting industry. Thanks to their bio-engineered liver and cornea tissue they are getting ahead of the game fast.

Pandorum is not the first tissue engineering startup that attempts to create a cornea, last October, North Carolina-based bioprinting startup Precise Bio announced plans to advance its research into bioprinted corneas. The company was the first to transplant a 3D printed cornea graft into an animal and plans to start with a human cornea suitable for transplantation soon. While earlier this year, Korean researchers successfully bioprinted tissue for cornea with transparent bioink. On the academic front, Newcastle University researchers successfully 3D printed human corneas from stem cell bioink. With all the startups and university researchers racing to create the most compatible and functional organs that could one day replace organ transplantation, the stakes are high with pressure from the public opinion, anxiously awaiting a change in the way medicine will solve some of the most complex illnesses. The 3D bioprinting field is entering a very exciting time, but some big challenges in bringing these therapies to reality are still out there.

Korean Researchers Successfully BioPrint Tissue for Corneas With Transparent Bioink

In ‘Characterization of cornea-specific bioink: high transparency, improved in vivo safety,’ researchers Hyeonji Kim, Moon-Nyeo Park, and Jisoo Kim explore the materials and processes surrounding corneal tissue engineering.

Patients waiting for cornea transplants are often in for a long wait due to a decrease in the supply to hospitals. And while artificial corneas have been created to solve this problem, they are challenging to implant because of restricted tissue integration. With the creation of a cornea-derived decellularized extracellular matrix (Co-dECM), the researchers endeavored to make further progress in corneal regeneration.

Their new bioink demonstrates similarities to the human cornea and offer the transparency required for human vision. Because this eye tissue is vital to being able to see, damage to the cornea becomes a serious issue and can even lead to blindness. The researchers cite data from the World Health Organization stating that around 285 million individuals in the US have visual deficiencies due to corneal disease. On average, a corneal transplant involves a wait time of 2,134 days, and out of all donor list waits, it is the longest.

“Moreover, the waiting time unfortunately has become even longer because of a shortage of donor cornea due to the rapid increase in the number of procedures for laser-based treatments and surgery (e.g., laser in-situ keratomileusis (LASIK)), which makes the cornea undonatable,” state the researchers. “To replace donor corneas, clinically available synthetic corneas are widely being used including Keratoprosthesis (KPro, made of poly(methyl methacrylate) (PMMA)), and AlphaCorTM(poly(2-hydroxyethyl methacrylate), PHEMA).”

Artificial corneas have been the cause of numerous and severe reactions though, unfortunately. This is due to adverse reactions as the body begins to reject the foreign objects. Researchers turned to bioengineering to create more hospitable corneas. They began by harvesting entire corneas from bovine eyeballs, procured from a Korean slaughterhouse. The corneas were further prepared in an extensive sterilization process and then stored.

Schematic of Co-dECM gel preparation and its validation.

Gel samples were made and then evaluated for growth of tissue. The team then used their in-house bioprinting system to fabricate Co-dECM bioink encapsulating cells. Experiments were performed on mice and rabbits to assess biocompatibility of the ink, with mice functioning to demonstrate immune responses. This was true in the rabbits, but the scientists also evaluated cell activity after samples were implanted (see the research paper regarding treatment according to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research).

Optical properties of Co-dECM gel. (a) Gross images (scale bar: 2 mm). (b) Light transmittance variations of 2% Co-dECM gel, 2% Col, and human cornea at different wavelengths of visible light spectrum. (c) SEM micrographs of samples (scale bar: 10 µm). (d) Thicknesses of collagen fibers for Co-dECM gel (Co-dECM 1X), Co-dECM gel mixed with Col (Co-dECM 0.5X), and Col (Co-dECM 0X). *p < 0.05, ***p < 0.005.

The researchers analyzed the following in the Co-dECM gel:

  • Transparency and microstructure
  • Internal biomolecular growth factors
  • Gene expression pattern

The two control groups were comprised of collagen hydrogel and native human cornea.

Transparency of the Co-dECM gel was rated ‘excellent,’ with the team stating such high marks could be due to ‘thin collagen fibrils’ associated with the graft.

 “…Co-dECM gel also contains various growth factors, including fibroblast growth factor (FGF), insulin-like growth factor (IGF), and transforming growth factor (TGF), which are abundantly observed in the native cornea, stated the researchers. “In addition, we verified the biological effects of Co-dECM from the differentiation of stem cells into keratocyte lineage by culturing hTMSCs in the Co-dECM gel.”

“The representative markers for cornea stromal layer, such as Keratocan (KERA), Aldehyde dehydrogenase (ALDH), were investigated after 14-day culture, expressed, respectively, as 7.30 and 11.97 times greater than the cells cultured in the Col gel. These results indicate that the Co-dECM bioink provides microstructural and biochemical cues for cells to induce them to differentiate into keratocyte lineage.”

H&E stained images using rabbit model. Optical micrographs, OCT images with H&E stained images on day 28, and the number of immune cells on days 14 and 28. Scale bar: 50 µm.

In testing further, the researchers 3D printed lattices. They maintained their structure after crosslinking, and tissue remained alive.

“This study demonstrated the feasibility of Co-dECM bioink applications for the fabrication of patient-specific shaped artificial corneas,” concluded the researchers. “Thus, the proposed Co-dECM bioink can be applied to 3D cell printing technique to provide cornea-mimicking microenvironments. It may support progress in the field of cornea tissue engineering in future applications.”

While there are plenty of 3D printing projects created and enjoyed with sheer whimsy, the innovations brought forth in the medical field offer serious potential for changing the lives of patients. Bioprinting offers myriad benefits to the medical field and is quickly attaining superstar status as an offshoot to the 3D printing realm. The engineering of tissue alone is a huge stride for science, but of course it is the eventual potential we all see it unlocking that has everyone excited, regarding the ability to 3D print the human organ. So far, scientists and medical professionals have used bioprinting to advance the study of Alzheimer’s, fabricate a lab-grown bladder, and also create orbital implants for intricate surgeries.

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[Source / Images: Characterization of cornea-specific bioink: high transparency, improved in vivo safety]