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Making Organs In Bioreactors : An Insight
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Ever imagined the production of fully-functional organs in Bioreactors? And, not only this! their successful transplantation in the target organism?! Well, if not, then the research by Dr. Harold C Ott of Harvard Medical School, Boston, Massachusetts, USA published in The Nature Medicine, on April 14, 2013, should not only make you imagine so, but believe too!

Dr. Harold's lab works on in-vitro synthesis of new organs for living organisms, but with a distinct approach. Rather than de-novo synthesis of the entire organ from the totipotent cells in animal cell-culture facility (which hasn't resulted in any fully-functional and organized organ till date), his approach is to use the target organism's organ itself as the base for initiating the organ-culture.

The Ideal Approach:
The organ to be changed is taken out of the organism. It is stripped off all the cells/tissue matter inside using a mild detergent solution (process called De-cellularization). What's left behind is a scaffold of the organ (sort of a mould, made of the basic connective tissue required for the structural organization of the organ). The scaffold is then supplied with the embryonic cells for the organ/endothelium from the young organism of preferably same species (preferably, a close relative). A pressure gradient is applied to the scaffold to ensure the retention of the cells in the scaffold. The scaffold is kept in a perfusion bioreactor with the requisite media and allowed to differentiate into a fully/partially functional organ. The organ is then transplanted into the target organism and checked for the functional characteristics. This is the way Harold's approach should work ideally in near future, as explained by Dr. Harold C Ott in this Nature Video!:


Following is the step wise insight into the actual research done by Harold and team on synthesizing a "kidney" in the lab in a bioreactor, which was later transplanted successfully in a rat.

1. Cadaveric (dead) Rat Kidneys were obtained.

2. They were de-cellularized by renal artery perfusion using mild SDS, deionized water and Triton X-100 solution (detergent) at a low constant pressure (around 40 mm Hg), so that the organ might not burst/collapse.

3. Scaffolding preserved the tissue architecture, but removed all cells/nuclei. Vascular, cortical and medullary architecture, a collecting system and ureters were retained in the scaffold.

4. Preservation of the basic architecture was very important as the key processes of filtration (glomerular basement membrane), secretion and reabsorption (tubular basement membrane) require the highly organized structural framework.

5. Washing with PBS was done to remove the traces of detergent.

6. Regeneration of kidney tissue was attempted by repopulating the scaffold with endothelial and epithelial cells. Human umbilical venous endothelial cells (HUVECs) and Rat neonatal kidney cells (NKCs) were perfused through the scaffold using 40cm H20 pressure gradient. Maintenance of the pressure was cruacial for retaining the cells in the scaffold, without damage/leaking of the cells.

7. The seeded scaffold was then transferred to a perfusion bioreactor having optimal organ-culture conditions and nutrient media.

8. HUVECs lining of the vasculature in the entire scaffold was observed in 3-5 days of bioreactor inoculation.

9. As the NKCs were obtained from neonatal rats, maturation signals such as glucocorticoids and catecholamines were supplied in the media for differentiation of the tubular apparatus to produce concentrated urine.
Histologic evaluation after within 4 days indicated epithelial and endothelial cells repopulated on the scaffold without any damage to the glomerular, tubular and vascular architecture. The optimal usage of the pressure gradient enabled proper engrafting of the NKCs and HUVECs at the vascular/epithelial compartments they were meant for in the normal organ. Nephrons' microanatomy was established in the culturing organ, laying the foundation for the basic functions of filtration, secretion, absorption and urine production. Non-specific grafting wasn't observed during immunostaining, which was a positive signal towards proper differentiation.

10. Overall, a 12 day culture resulted in partially functional organ.

11. After the culture, Orthotopic (at the normal place, where it's meant to be) transplantation of the lab-generated kidney was done. No-bleeding/blood leakage was observed in the vasculature of the regenerated kidney. Urine was continously produced with clearance of metabolites till experimental observation without any clotting either.

12. The quality of urine matched the quality produced during in-vitro observations (with glucose, albumins, urea, creatinine etc in same concentration as in-vitro, though much higher than the actual physiological requirement).

Conclusion:
Though, lot needs to be done, this approach of Dr. Harold and team, has highlighted an altogether a new way and hope for meeting the needs of millions of organ transplant seekers across the globe (especially the kidney transplant seekers). Considering the immediate functioning of the regenerated kidneys after transplant, this approach seems highly promising, especially considering the fact that even 'cadaveric' kidneys can act as the source of the scaffold. Optimization of cell seeding strategies, isolation, differentiation and expansion of the required cell types from clinically feasible sources and upscaling of the biomimetic organ culture is what Dr. Harold's team believes may pave the way for in-vitro production of fully-functional organs!

Harold's research is not limited to a particular organ, rather his team has been working on a lot of organs including heart and lungs (as you might have seen in the video). In fact, this research was motivated by the successful attempts in scaffolding of heart and lungs ECM by his (and other) teams. So, let's hope that this research treads fast on the right path towards providing a radical new way of bringing an end to the long wait of the patients looking for an organ donor and save his/her life!

Thanks
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