INJECTABLE, CELL-RESPONSIVE SUPRAMOLECULAR HYDROGEL FOR LUNG ORGANOID GRAFTS

June Y. Park1,2, Kelly Evans2, Stefan Mommer1, Oren A. Scherman1 , Joo H. Lee2

University of Cambridge, UK1Melville Laboratory for Polymer Synthesis, Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, UK.2Cambridge Institute for Medical Research and Wellcome Trust/MRC Stem Cell Institute, University of Cambridge, Cambridge, UK.

Organoids, or 3D stem cell derived organ-like structures, are emerging as a potential transplant replacement1, 2, 3, 4 but local,minimally invasive delivery remains a challenge5. An injectable, cell-responsive supramolecular hydrogel transiently crosslinkedwith cucurbit[8]uril-mediated host guest chemistry is developed for potential treatment of chronic, aging related lung diseasessuch as idiopathic pulmonary fibrosis (IPF). These preliminary results confirm the shear thinning and self healing mechanicalproperties of the hydrogel, as well as capacity to promote primary stem cell organoid growth in 3D culture.

OverviewEngrafting organoids, or 3D stem-cell-derived structures thatresemble in-vivo organs, shows promising results asregenerative therapy.1, 2, 3, 4, but highly controlled, localdelivery remains a challenge5.

Hydrogel SynthesisHyaluronic acid (HA) was functionalized with methacrylicanhydride at room temperature overnight in the dark. Cell-responsive peptides were synthesized using microwavepeptide synthesizer. Peptides were added to methacrylate vianucleophilic addition. A hydrogel was formed upon theaddition of the cucurbit[8]uril (CB[8]) macrocycle whichcreated dynamic links between adjacent polymer chains.

Organoid CultureLung organoids can be formed from different types of adultstem cells. Figure 3 shows the typical images of lungorganoids grown from various types of lung stem cells.

Figure 2. Synthesis of functionalized polymer with cleavable peptide sequence

Figure 1. Schematic of supramolecular hydrogel formation and lung organoid graft

CB[8]+

Organoid

+

Gradual degradation of scaffoldwith organoid growth

Organoid-loaded hydrogelinjected into distal lung

Primary stem cell viability and organoids forming capacitywere demonstrated via image captured using fluorescencemicroscopy on Day 10.

Transwell Insert

Upper compartment

Hydrogel with organoids

Microporous membrane

Lower compartment

Culture ConditionDay 10 Mouse AlveolarOrganoids

Figure 4. Culture Condition and image of organoid grown in hydrogel on Day 10

1. Rossi, G., Manfrin, A., & Lutolf, M. P. (2018). Progress and potential in organoidresearch. Nature Reviews Genetics, 19(11), 671–687.

2. Clevers, H. (2016). Modeling Development and Disease with Organoids. Cell, 165(7),1586–1597. https://doi.org/10.1016/J.CELL.2016.05.082

3. Murry, C. E., & Keller, G. (2008). Differentiation of Embryonic Stem Cells to ClinicallyRelevant Populations: Lessons from Embryonic Development. Cell, 132(4), 661–680.https://doi.org/10.1016/J.CELL.2008.02.008

4. Rossi, G., Manfrin, A., & Lutolf, M. P. (2018). Progress and potential in organoidresearch. Nature Reviews Genetics, 19(11), 671–687.https://doi.org/10.1038/s41576-018-0051-9

5. Huch, M., Knoblich, J. A., Lutolf, M. P., & Martinez-Arias, A. (2017). The hope and thehype of organoid research. Development (Cambridge, England), 144(6), 938–941.https://doi.org/10.1242/dev.150201

+

Methacrylic anhydride

MMP CleavableGuest

RGD

Pro-Leu Gly-Met-Trp-Ser-ArgCys Phe

Arg-Gly-Asp SerCys

Hyaluronic AcidConclusionWith the growing number of stem cell organoid research fortreatment of diseases and cell therapies in the market, themode of cell therapy delivery will become increasinglyimportant. Using injectable, cell-responsive supramolecularpolymer hydrogel, this project aims to pave a path towards aminimally invasive regenerative medicine for aging-relateddiseases.

Airw

ay

Airw

ay/A

lve

oli

Alv

eo

lar

SP

C

T1a

DA

PI

CC

SP

Acety

l-tub

DA

PI

CC

SP

SP

CD

AP

I

Lee et al. Cell. 2014

Figure 5. Hypothetical schematic of organoid-based regenerative therapy

Figure 3. Airway and alveolar lineage differentiation