Methods for controlled induction of 3D cylindrical neuroepithelial tubes
Described herein are methods, compositions, and kits for forming engineered in vitro biomimetic, three-dimensional, tubular organoid structures by directed differentiation of human pluripotent stem cells within tubular channels formed in a hydrogel.
1. A method of preparing an engineered biomimetic 3 D organoid in vitro, the method comprising:
(a) providing a hydrogel having a channel therein, the channel having a first end and a second end;
(b) sealing one or more of the first and second ends of the channel;
(c) seeding the channel with human pluripotent stem cells (hPSCs),
(d) contacting the hydrogel comprising the hPSC-seeded channel to an alginate solution, and then contacting the alginate-contacted hydrogel to a divalent cation solution, thereby producing a hPSC-seeded channel encapsulated in crosslinked hydrogel, and
(e) culturing the encapsulated hPSC-seeded channel in a culture medium for about four (4) to about sixteen (16) days for the hPSCs within the channel to differentiate into neuroepithelial cells, whereby a biomimetic 3D organoid comprising polarized neuroepithelial cells and having microscale cellular organization similar to that of an in vivo developing human neural tube is obtained.
2. The method of claim 1 , wherein the hydrogel is an alginate hydrogel.
3. The method of claim 1 , wherein the divalent cation solution is a calcium chloride (CaCl 2 ) solution.
4. The method of claim 1 , wherein sealing comprises contacting the first end or the second end of the channel to an alginate solution and contacting the alginate-contacted end to a divalent cation solution, thereby producing a channel comprising a sealed end and an unsealed end.
5. The method of claim 4 , wherein seeding the channel comprises injecting a hPSC suspension having a cell density of about 500,000 cells/μl or less into the unsealed end.
6. The method of claim 1 , wherein sealing comprises contacting the first end and the second end of the channel into an alginate solution and contacting each alginate- contacted end into a divalent cation solution, thereby producing a channel comprising a first sealed end and a second sealed end.
7. The method of claim 6 , wherein seeding the channel comprises injecting a hPSC suspension having a cell density of about 500,000 cells/μl or less into the first sealed end or the second sealed end.
8. The method of claim 7 , further comprising re-sealing the end into which the hPSC suspension is injected, wherein re-sealing comprises;
contacting an alginate solution to the hPSC suspension-injected end; and
contacting the alginate-contacted end to a divalent cation solution to cross-link the alginate, thereby producing a hPSC-seeded channel comprising a sealed end and a re-sealed end.
9. The method of claim 1 , wherein the biomimetic 3D organoid is a biomimetic neuroepithelial organoid comprising polarized neural stem cells.
10. The method of claim 1 , wherein the channel is substantially tubular.
11. The method of claim 10 , wherein the diameter of the substantially tubular channel is between about 50 μm to about 700 μm.
12. The method of claim 10 , wherein the diameter of the substantially tubular channel is between about 100 μm to about 300 μm.
13. The method of claim 1 , wherein the culture medium is sufficient to promote the self-organization and spontaneous morphogenesis of the hPSCs into neuroepithelial organoids.
14. The method of claim 13 , wherein the culture medium is a neural differentiation medium comprising water, salts, amino acids, vitamins, a carbon source, a buffering agent, selenium, ascorbate, insulin, transferrin, and a Rho kinase (ROCK) inhibitor.
15. The method of claim 14 , wherein the hydrogel comprising the cell-seeded channel is cultured within a spinner flask containing the neural differentiation medium.
16. The method of claim 1 , wherein the hydrogel is produced by the method comprising:
(a) providing a sacrificial template of a predefined shape immobilized within a casting chamber,
(b) introducing into the casting chamber a volume of hydrogel polymer solution sufficient to surround the sacrificial template,
(c) contacting the hydrogel polymer with a cross-linking solution to form a hydrogel shell surrounding the sacrificial template, and
(d) removing the sacrificial template, thereby providing a hydrogel with a channel therein.
17. The method of claim 16 , wherein the sacrificial template is a water soluble thermoplastic-divalent cationic salt composite material.
18. The method of claim 16 , wherein the sacrificial template is poly(vinyl alcohol) material coated in divalent cations.