Screening hydrogels for antifibrotic properties by implanting cellularly barcoded alginates in mice and a non

Aug 23, 2023

Nature Biomedical Engineering volume 7, pages 867–886 (2023)Cite this article

3007 Accesses

1 Citations

118 Altmetric

Metrics details

Screening implantable biomaterials for antifibrotic properties is constrained by the need for in vivo testing. Here we show that the throughput of in vivo screening can be increased by cellularly barcoding a chemically modified combinatorial library of hydrogel formulations. The method involves the implantation of a mixture of alginate formulations, each barcoded with human umbilical vein endothelial cells from different donors, and the association of the identity and performance of each formulation by genotyping single nucleotide polymorphisms of the cells via next-generation sequencing. We used the method to screen 20 alginate formulations in a single mouse and 100 alginate formulations in a single non-human primate, and identified three lead hydrogel formulations with antifibrotic properties. Encapsulating human islets with one of the formulations led to long-term glycaemic control in a mouse model of diabetes, and coating medical-grade catheters with the other two formulations prevented fibrotic overgrowth. High-throughput screening of barcoded biomaterials in vivo may help identify formulations that enhance the long-term performance of medical devices and of biomaterial-encapsulated therapeutic cells.

This is a preview of subscription content, access via your institution

Access Nature and 54 other Nature Portfolio journals

Get Nature+, our best-value online-access subscription

$29.99 / 30 days

cancel any time

Subscribe to this journal

Receive 12 digital issues and online access to articles

$79.00 per year

only $6.58 per issue

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

The main data supporting the results in this study are available within the paper and its Supplementary Information. The raw sequencing data generated in this study can be accessed via Figshare at https://figshare.com/projects/In_vivo_screening_of_hydrogel_library_using_cellular_barcoding_identifies_biomaterials_that_mitigate_host_immune_responses_and_fibrosis/144375. The datasets generated and analysed during the study are available for research purposes from the corresponding authors on reasonable request.

Anderson, J. M., Rodriguez, A. & Chang, D. T. Foreign body reaction to biomaterials. Semin. Immunol. 20, 86–100 (2008).

CAS PubMed Google Scholar

Wick, G. et al. The immunology of fibrosis. Annu. Rev. Immunol. 31, 107–135 (2013).

CAS PubMed Google Scholar

Wynn, T. A. & Ramalingam, T. R. Mechanisms of fibrosis: therapeutic translation for fibrotic disease. Nat. Med. 18, 1028–1040 (2012).

CAS PubMed PubMed Central Google Scholar

Veiseh, O. & Vegas, A. J. Domesticating the foreign body response: recent advances and applications. Adv. Drug Deliv. Rev. 144, 148–161 (2019).

CAS PubMed PubMed Central Google Scholar

Kenneth Ward, W. A review of the foreign-body response to subcutaneously-implanted devices: the role of macrophages and cytokines in biofouling and fibrosis. J. Diabetes Sci. Technol. 2, 768–777 (2008).

CAS PubMed PubMed Central Google Scholar

Vegas, A. J. et al. Combinatorial hydrogel library enables identification of materials that mitigate the foreign body response in primates. Nat. Biotechnol. 34, 345–352 (2016).

CAS PubMed PubMed Central Google Scholar

Veiseh, O. et al. Size- and shape-dependent foreign body immune response to materials implanted in rodents and non-human primates. Nat. Mater. 14, 643–651 (2015).

CAS PubMed PubMed Central Google Scholar

Darnell, M. & Mooney, D. J. Leveraging advances in biology to design biomaterials. Nat. Mater. 16, 1178–1185 (2017).

CAS PubMed Google Scholar

Peters, A., Brey, D. M. & Burdick, J. A. High-throughput and combinatorial technologies for tissue engineering applications. Tissue Eng. B 15, 225–239 (2009).

CAS Google Scholar

Grainger, D. W. All charged up about implanted biomaterials. Nat. Biotechnol. 31, 507–509 (2013).

CAS PubMed Google Scholar

Zhang, L. et al. Zwitterionic hydrogels implanted in mice resist the foreign-body reaction. Nat. Biotechnol. 31, 553–556 (2013).

CAS PubMed Google Scholar

Witherel, C. E., Abebayehu, D., Barker, T. H. & Spiller, K. L. Macrophage and fibroblast interactions in biomaterial-mediated fibrosis. Adv. Healthc. Mater. 8, e1801451 (2019).

PubMed PubMed Central Google Scholar

Ratner, B. D. Biomaterials: been there, done that, and evolving into the future. Annu. Rev. Biomed. Eng. 21, 171–191 (2019).

CAS PubMed Google Scholar

Bank, R. A. Limiting biomaterial fibrosis. Nat. Mater. 18, 781 (2019).

CAS PubMed Google Scholar

Noskovicova, N. et al. Suppression of the fibrotic encapsulation of silicone implants by inhibiting the mechanical activation of pro-fibrotic TGF-β. Nat. Biomed. Eng. https://doi.org/10.1038/s41551-021-00722-z (2021).

Ernst, A. U., Wang, L.-H. & Ma, M. Islet encapsulation. J. Mater. Chem. B 6, 6705–6722 (2018).

CAS PubMed Google Scholar

Kearney, C. J. & Mooney, D. J. Macroscale delivery systems for molecular and cellular payloads. Nat. Mater. 12, 1004–1017 (2013).

CAS PubMed Google Scholar

Bochenek, M. A. et al. Alginate encapsulation as long-term immune protection of allogeneic pancreatic islet cells transplanted into the omental bursa of macaques. Nat. Biomed. Eng. 2, 810–821 (2018).

CAS PubMed PubMed Central Google Scholar

Vegas, A. J. et al. Long-term glycemic control using polymer-encapsulated human stem cell-derived beta cells in immune-competent mice. Nat. Med. 22, 306–311 (2016).

Bidarra, S. J., Barrias, C. C. & Granja, P. L. Injectable alginate hydrogels for cell delivery in tissue engineering. Acta Biomater. 10, 1646–1662 (2014).

CAS PubMed Google Scholar

Zhao, L., Weir, M. D. & Xu, H. H. K. An injectable calcium phosphate-alginate hydrogel-umbilical cord mesenchymal stem cell paste for bone tissue engineering. Biomaterials 31, 6502–6510 (2010).

CAS PubMed PubMed Central Google Scholar

Wang, X. et al. A nanofibrous encapsulation device for safe delivery of insulin-producing cells to treat type 1 diabetes. Sci. Transl. Med. 13, eabb4601 (2021).

CAS PubMed PubMed Central Google Scholar

Xie, X. et al. Reduction of measurement noise in a continuous glucose monitor by coating the sensor with a zwitterionic polymer. Nat. Biomed. Eng. 2, 894–906 (2018).

CAS PubMed PubMed Central Google Scholar

Jacobs-Tulleneers-Thevissen, D. et al. Sustained function of alginate-encapsulated human islet cell implants in the peritoneal cavity of mice leading to a pilot study in a type 1 diabetic patient. Diabetologia 56, 1605–1614 (2013).

CAS PubMed Google Scholar

Robitaille, R. et al. Inflammatory response to peritoneal implantation of alginate-poly-L-lysine microcapsules. Biomaterials 26, 4119–4127 (2005).

CAS PubMed Google Scholar

Rokstad, A. M. et al. Alginate microbeads are complement compatible, in contrast to polycation containing microcapsules, as revealed in a human whole blood model. Acta Biomater. 7, 2566–2578 (2011).

CAS PubMed Google Scholar

King, A., Sandler, S. & Andersson, A. The effect of host factors and capsule composition on the cellular overgrowth on implanted alginate capsules. J. Biomed. Mater. Res. 57, 374–383 (2001).

CAS PubMed Google Scholar

Manoury, B., Caulet-Maugendre, S., Guenon, I., Lagente, V. & Boichot, E. TIMP-1 is a key factor of fibrogenic response to bleomycin in mouse lung. Int. J. Immunopathol. Pharmacol. 19, 471–487 (2006).

CAS PubMed Google Scholar

Desai, T. & Shea, L. D. Advances in islet encapsulation technologies. Nat. Rev. Drug Discov. 16, 338–350 (2017).

CAS PubMed Google Scholar

Tuch, B. E. et al. Safety and viability of microencapsulated human islets transplanted into diabetic humans. Diabetes Care 32, 1887–1889 (2009).

CAS PubMed PubMed Central Google Scholar

Bose, S. et al. A retrievable implant for the long-term encapsulation and survival of therapeutic xenogeneic cells. Nat. Biomed. Eng. 4, 814–826 (2020).

CAS PubMed PubMed Central Google Scholar

Farah, S. et al. Long-term implant fibrosis prevention in rodents and non-human primates using crystallized drug formulations. Nat. Mater. 18, 892–904 (2019).

CAS PubMed PubMed Central Google Scholar

Krutzik, P. O. & Nolan, G. P. Fluorescent cell barcoding in flow cytometry allows high-throughput drug screening and signaling profiling. Nat. Methods 3, 361–368 (2006).

CAS PubMed Google Scholar

Strobel, B. et al. High-throughput identification of synthetic riboswitches by barcode-free amplicon-sequencing in human cells. Nat. Commun. 11, 714 (2020).

CAS PubMed PubMed Central Google Scholar

Lyons, E., Sheridan, P., Tremmel, G., Miyano, S. & Sugano, S. Large-scale DNA barcode library generation for biomolecule identification in high-throughput screens. Sci. Rep. 7, 13899 (2017).

PubMed PubMed Central Google Scholar

Binan, L., Drobetsky, E. A. & Costantino, S. Exploiting molecular barcodes in high-throughput cellular assays. SLAS Technol. 24, 298–307 (2019).

CAS PubMed Google Scholar

Bykov, Y. S. et al. High-throughput ultrastructure screening using electron microscopy and fluorescent barcoding. J. Cell Biol. 218, 2797–2811 (2019).

CAS PubMed PubMed Central Google Scholar

Oliveira, M. B. & Mano, J. F. High-throughput screening for integrative biomaterials design: exploring advances and new trends. Trends Biotechnol. 32, 627–636 (2014).

CAS PubMed Google Scholar

Bratlie, K. M. et al. Rapid biocompatibility analysis of materials via in vivo fluorescence imaging of mouse models. PLoS ONE 5, e10032 (2010).

PubMed PubMed Central Google Scholar

Chatterjee, K. et al. The effect of 3D hydrogel scaffold modulus on osteoblast differentiation and mineralization revealed by combinatorial screening. Biomaterials 31, 5051–5062 (2010).

CAS PubMed PubMed Central Google Scholar

Vega, S. L. et al. Combinatorial hydrogels with biochemical gradients for screening 3D cellular microenvironments. Nat. Commun. 9, 614 (2018).

PubMed PubMed Central Google Scholar

Andreiuk, B. et al. Fluorescent polymer nanoparticles for cell barcoding in vitro and in vivo. Small 13, 1701582 (2017).

Google Scholar

Lokugamage, M. P., Sago, C. D. & Dahlman, J. E. Testing thousands of nanoparticles in vivo using DNA barcodes. Curr. Opin. Biomed. Eng. 7, 1–8 (2018).

PubMed PubMed Central Google Scholar

Paunovska, K. et al. A direct comparison of in vitro and in vivo nucleic acid delivery mediated by hundreds of nanoparticles reveals a weak correlation. Nano Lett. 18, 2148–2157 (2018).

CAS PubMed PubMed Central Google Scholar

Vorholt, S. M. et al. High-throughput screening of blood donors for twelve human platelet antigen systems using next-generation sequencing reveals detection of rare polymorphisms and two novel protein-changing variants. Transfus. Med. Hemother. 47, 33–44 (2020).

PubMed PubMed Central Google Scholar

Nyyssonen, M. et al. Coupled high-throughput functional screening and next generation sequencing for identification of plant polymer decomposing enzymes in metagenomic libraries. Front. Microbiol. 4, 282 (2013).

PubMed PubMed Central Google Scholar

Klaper, R., Arndt, D., Bozich, J. & Dominguez, G. Molecular interactions of nanomaterials and organisms: defining biomarkers for toxicity and high-throughput screening using traditional and next-generation sequencing approaches. Analyst 139, 882–895 (2014).

CAS PubMed Google Scholar

Ishikawa, F. et al. Development of functional human blood and immune systems in NOD/SCID/IL2 receptor γ chainnull mice. Blood 106, 1565–1573 (2005).

Shultz, L. D. et al. Human lymphoid and myeloid cell development in NOD/LtSz-scid IL2Rγnull mice engrafted with mobilized human hemopoietic stem cells. J. Immunol. 174, 6477–6489 (2005).

Doloff, J. C. et al. Colony stimulating factor-1 receptor is a central component of the foreign body response to biomaterial implants in rodents and non-human primates. Nat. Mater. 16, 671–680 (2017).

CAS PubMed PubMed Central Google Scholar

Doloff, J. C. et al. The surface topography of silicone breast implants mediates the foreign body response in mice, rabbits and humans. Nat. Biomed. Eng. 5, 1115–1130 (2021).

CAS PubMed Google Scholar

Qi, M. et al. Survival of human islets in microbeads containing high guluronic acid alginate crosslinked with Ca2+ and Ba2+. Xenotransplantation 19, 355–364 (2012).

PubMed PubMed Central Google Scholar

Download references

This work was supported by the US National Institutes of Health (R01 DK120459 to O.V. and D.Y.Z), JDRF (3-SRA-2021-1023-S-B to O.V.), and the Rice University Academy Fellowship (to M.I.J.). We thank the support of the National Science Foundation for access to the ToF-SIMS, supported through CBET1626418. ToF-SIMS analyses and spin coating were carried out with support provided by the Shared Equipment Authority at Rice University. We also thank Rice University animal resource facility staff for their assistance with animal research.

Sudip Mukherjee

Present address: School of Biomedical Engineering, Indian Institute of Technology (BHU), Varanasi, Uttar Pradesh, India

David Yu Zhang

Present address: NuProbe USA, Houston, TX, USA

These authors contributed equally: Sudip Mukherjee, Boram Kim, Lauren Y. Cheng.

Department of Bioengineering, Rice University, Houston, TX, USA

Sudip Mukherjee, Boram Kim, Lauren Y. Cheng, Michael David Doerfert, Jiaming Li, Andrea Hernandez, Lily Liang, Maria I. Jarvis, Cody Fell, Ping Song, David Yu Zhang & Omid Veiseh

CellTrans, Inc., Chicago, IL, USA

Peter D. Rios, Sofia Ghani, Ira Joshi & Douglas Isa

Department of Biomedical Engineering, Cornell University, Ithaca, NY, USA

Trisha Ray

SIMS Laboratory, Shared Equipment Authority, Rice University, Houston, TX, USA

Tanguy Terlier

Department of Hematopathology, Division of Pathology/Lab Medicine, The University of Texas MD Anderson Cancer Center, Houston, TX, USA

Roberto N. Miranda

Division of Transplant Surgery, University of Virginia, Charlottesville, VA, USA

Jose Oberholzer

You can also search for this author in PubMed Google Scholar

You can also search for this author in PubMed Google Scholar

You can also search for this author in PubMed Google Scholar

You can also search for this author in PubMed Google Scholar

You can also search for this author in PubMed Google Scholar

You can also search for this author in PubMed Google Scholar

You can also search for this author in PubMed Google Scholar

You can also search for this author in PubMed Google Scholar

You can also search for this author in PubMed Google Scholar

You can also search for this author in PubMed Google Scholar

You can also search for this author in PubMed Google Scholar

You can also search for this author in PubMed Google Scholar

You can also search for this author in PubMed Google Scholar

You can also search for this author in PubMed Google Scholar

You can also search for this author in PubMed Google Scholar

You can also search for this author in PubMed Google Scholar

You can also search for this author in PubMed Google Scholar

You can also search for this author in PubMed Google Scholar

You can also search for this author in PubMed Google Scholar

You can also search for this author in PubMed Google Scholar

S.M., B.K., L.Y.C., D.Y.Z. and O.V. designed the studies, analysed data and wrote the paper. S.M., B.K., L.Y.C., M.D.D., J.L., A.H., L.L., M.I.J., P.D.R., S.G., I.J., D.I., T.R., T.T., C.F., P.S., J.O., O.V. and D.Y.Z. conducted the experiments. S.M., B.K., M.D.D. and L.Y.C. carried out the statistical analyses and prepared displays communicating datasets. R.N.M., T.T., D.Y.Z. and O.V. provided advice and technical support throughout, and D.Y.Z. and O.V. supevised the study. All authors discussed the results and the preparation of the paper.

Correspondence to David Yu Zhang or Omid Veiseh.

O.V. is cofounder and significant shareholder of Sigilon Therapeutics, Avenge Bio and Pana Bio. D.Y.Z. owns significant equity in and receives consulting income from NuProbe Global and Torus Biosystems, and owns significant equity in Pana Bio. J.O. is a founding scientist and significant shareholder of Sigilon Therapeutics and CellTrans Inc.

Nature Biomedical Engineering thanks the anonymous reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Left of the scheme are two of the previous triazole-containing lead alginates (B1-A21 and Z1-A34) that prevent fibrosis in mouse and NHP models. A total of 211 new alginate analogs were synthesized by varying the lead hydrophilic linker (Azido PEG-amine) and hydrophobic linker (iodobenzylamine) combined with a set of alkyne classes highlighted in blue colors.

a, Gelation assay of alginate analogs using Rhodamine B entrapment. b, Representative images of capsule formation using the alginate analogs. Scale bar, 2 mm. c, After initial characterization studies, including purity, solubility, and gel-forming ability, 149 alginate polymers were used for the screening test. d, Mechanical testing (work to burst) with different alginate formulations. One-way ANOVA with Bonferroni correction was used for statistical analysis; ***P = 0.0007 (Z1-A34), ***P = 0.0009 (B1-A51), ns; non significant for comparisons with SLG20. All error bars denote mean ± s.e.m of n = 20 replicates. e-i, FITC-dextran permeability test with different modified alginates. All error bars denote mean ± s.e.m of n = 5 replicates.

The library preparation involved a multiplex PCR step in amplifying the SNP loci, a barcoding PCR step to add position barcode to each sample, and a ligation-based sequencing adapter amendment procedure. a, Before optimization, the library on-target rate was <10%, with primer-dimers and non-specific PCR products contributing to the majority of reads. b, After optimization, the on-target rate was increased to >80% regardless of low DNA input (<1 ng) in the starting material.

Fastq NGS data was demultiplexed by row and column barcodes to re-group sequences amplified from the same DNA input. Then for each amplicon sequence, the grep function was applied to search the dominant and variant alleles to calculate variant allele frequency (VAF) for each SNP locus. If the encapsulated cells comprised only one donor, the VAF profile was compared against profiles of the 20 pre-screened HUVEC donors. The donor with the highest match rate was identified as the encapsulated donor cell. When one or two donors were used as encapsulated cells, the log-likelihood of all possible donor compositions was calculated. The composition with the highest overall log-likelihood was determined as the cell composition (Quality control for log-likelihood analysis: 1) at least 25/30 SNP loci had sequencing coverage >50; and 2) overall log-likelihood higher than -200, and 3) goodness measurement higher than 10 where goodness is defined as the difference of log-likelihood between the most likely and the second most likely donor pairs). The material corresponding to the identified donor cell or cell composition would be the material encapsulating cells.

a, Three different materials were tested; UP-VLVG (control), B1-A51 (one of the negative materials), and Z1-A34 (one of the positive materials). b, Schematic workflow of three materials screening containing mixed dual donors. c-d, After two weeks of implant, capsules were retrieved from each mouse (M1-M3) and were separated into three groups depending on fibrosis levels. e, Representative heatmap result of identified donor pair. f, 39 mapped to Z1-A34 (positive control material), coded by H16:H14 at 1:2 ratio; 4 mapped to UP-VLVG coded by H6:H8 at 1:2 ratio; 0 sample mapped to B1-A51. Overalled 43/45 samples were mapped from the 400 donor SNP profile. Proportions of each material corresponded to donor pairs were plotted, and Z1-A34 showed the highest value indicating the best immune-protective properties.

a, Capsules containing human islets were fabricated at final cell density with 4,000, 8,000, and 16,000 IEQ/alginate volume (mL). The final IEQ values in each capsule were 10, 20, and 40 IEQ per capsule, respectively. In each group, 500 µL, 250 µL, and 125 µL of capsules were implanted in IP space, containing total 2,000 IEQ per mouse. All error bars denote mean ± s.e.m of n = 20 biological replicates. b, Representative images of pre-implant Z1-A34 capsules. Dithizone staining indicates viable islets within the capsule matrix. After encapsulation, islets show good viability (live: green, dead: red).

a-b, The total intensity of two main peaks analyzed by ToF-SIMS (a, CN− and b, Br−) were plotted to compare with the unmodified catheter. c, XPS data for unmodified, Met-Z1A3, and Met-B2-A17 modified catheters showing wt. % of small molecule-specific atoms, indicating successful coating. d, Representative SEM images of unmodified and coated catheters. e-g, Surface characteristic of catheters coated with lead molecules using Raman spectroscopy. Pre-implant or post-explant catheters (4 or 8 weeks) were analyzed to confirm the presence of coated molecules (e, Met-Z1-A3, f, Met-B2-A17, g, Met-Z1-A34 catheters). All error bars denote mean ± s.e.m of n = 2 replicates.

Supplementary results, figures and tables.

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and Permissions

Mukherjee, S., Kim, B., Cheng, L.Y. et al. Screening hydrogels for antifibrotic properties by implanting cellularly barcoded alginates in mice and a non-human primate. Nat. Biomed. Eng 7, 867–886 (2023). https://doi.org/10.1038/s41551-023-01016-2

Download citation

Received: 23 February 2022

Accepted: 27 February 2023

Published: 27 April 2023

Issue Date: July 2023

DOI: https://doi.org/10.1038/s41551-023-01016-2

Anyone you share the following link with will be able to read this content:

Sorry, a shareable link is not currently available for this article.

Provided by the Springer Nature SharedIt content-sharing initiative