Fabrication of 3‐D Reconstituted Organoid Arrays by DNA‐Programmed Assembly of Cells (DPAC)

Michael E. Todhunter1, Robert J. Weber2, Justin Farlow1, Noel Y. Jee2, Alec E. Cerchiari3, Zev J. Gartner4

1 Tetrad Graduate Program, University of California, San Francisco, California, 2 Chemistry & Chemical Biology Graduate Program, University of California, San Francisco, California, 3 Graduate Program in Bioengineering, University of California, Berkeley, and University of California, San Francisco, California, 4 Center for Systems and Synthetic Biology, University of California, San Francisco, California
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/cpch.8
Online Posting Date:  September, 2016
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Abstract

Tissues are the organizational units of function in metazoan organisms. Tissues comprise an assortment of cellular building blocks, soluble factors, and extracellular matrix (ECM) composed into specific three‐dimensional (3‐D) structures. The capacity to reconstitute tissues in vitro with the structural complexity observed in vivo is key to understanding processes such as morphogenesis, homeostasis, and disease. In this article, we describe DNA‐programmed assembly of cells (DPAC), a method to fabricate viable, functional arrays of organoid‐like tissues within 3‐D ECM gels. In DPAC, dissociated cells are chemically functionalized with degradable oligonucleotide “Velcro,” allowing rapid, specific, and reversible cell adhesion to a two‐dimensional (2‐D) template patterned with complementary DNA. An iterative assembly process builds up organoids, layer‐by‐layer, from this initial 2‐D template and into the third dimension. Cleavage of the DNA releases the completed array of tissues that are captured and fully embedded in ECM gels for culture and observation. DPAC controls the size, shape, composition, and spatial heterogeneity of organoids and permits positioning of constituent cells with single‐cell resolution even within cultures several centimeters long. © 2016 by John Wiley & Sons, Inc.

Keywords: tissue array; DNA; synthetic biology; cell‐cell interactions; tissue engineering; patterning; organotypic; organoid; 3‐D culture

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Patterning Aqueous DNA Using Microscale Direct Writing
  • Basic Protocol 2: Passivation of Patterned Slides
  • Basic Protocol 3: DNA Labeling of Mammalian Cells
  • Support Protocol 1: Synthesis of Fatty‐Acid DNA
  • Basic Protocol 4: DNA‐Programmed Assembly of Cells on Patterned Glass
  • Support Protocol 2: Quantification of Cell‐DNA Functionalization
  • Support Protocol 3: Measurement of Surface‐DNA Functionalization
  • Support Protocol 4: Regeneration of DNA‐Patterned Surfaces
  • Support Protocol 5: Preparation of PDMS Flow Cells
  • Support Protocol 6: Construction of Mounted Toggle Clamp
  • Basic Protocol 5: 3‐D Transfer into Matrigel
  • Alternate Protocol 1: 3‐D Transfer into Agarose
  • Alternate Protocol 2: 3‐D Transfer into Collagen/Matrigel Mixtures
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Patterning Aqueous DNA Using Microscale Direct Writing

  Materials
  • 2 mM 20‐mer 5′‐amine‐DNA in distilled H 2O (available, e.g., from Eurofins MWG Operon; also see Strategic Planning)
  • 4× spotting buffer (see recipe)
  • UV/ozone apparatus (e.g., BioForce ProCleaner)
  • Aldehyde‐silanized glass slide (e.g., Nexterion AL, Applied Microarrays cat. no. 1064874)
  • Diamond scribe (Fisher, cat. no. 17‐467‐634)
  • Polymer forceps (BioForce Nanosciences, cat. no. PF‐Blunt)
  • Surface patterning tool (SPT; BioForce Nanosciences cat. no. SPT‐S‐C30S)
  • Microscale direct writing apparatus: BioForce Nano eNabler
  • 120ºC oven
  • Vacuum desiccator

Basic Protocol 2: Passivation of Patterned Slides

  Materials
  • Sodium borohydride (Fisher, cat. no. AC41947‐1000)
  • Absolute ethanol (Fisher, cat. no. BP2818‐4)
  • Phosphate‐buffered saline (e.g., Fisher, cat. no. R58190001A)
  • Patterned slides ( protocol 1)
  • 0.1% (w/v) sodium dodecyl sulfate (SDS) solution (store indefinitely at room temperature)
  • Dichloromethane (Fisher, cat. no. AC40692‐0040)
  • (Tridecafluoro‐1,1,2,2‐tetrahydrooctyl)dimethylchlorosilane (Gelest, cat. no. SIT8170.0)
  • Triethylamine (Fisher, cat. no. O4884‐100)
  • Glacial acetic acid (Fisher, cat. no. BP1185‐500)
  • 15‐cm Petri dishes
  • Orbital shaker
  • All‐glass Coplin jar (Fisher, cat. no. E94)
  • 1‐ml disposable plastic syringes)
  • 50‐ml conical tubes (e.g., Corning Falcon)
  • Metal tweezers (Fisher, cat. no. 09‐753‐50)
  • Vacuum desiccator

Basic Protocol 3: DNA Labeling of Mammalian Cells

  Materials
  • Cells (1 × 106 per reaction)
  • 5′‐Fatty‐acid DNA (adhesion Strand; see protocol 4)
  • 3′‐Fatty‐acid DNA (co‐anchor Strand; see protocol 4)
  • Sterile calcium‐and‐magnesium‐free Dulbecco's PBS (CMF‐DPBS; Fisher, cat. no. BW17512F12)
  • Flow buffer (see recipe)
  • Additional reagents and equipment for cell culture including preparing a single‐cell (monodisperse) suspension and counting cells (Phelan and May, )

Support Protocol 1: Synthesis of Fatty‐Acid DNA

  Materials
  • FMOC‐protected 3′ amino‐modified CPG, 1000 Å (Glen Research, cat. no. 20‐2958‐10)
  • 20% piperidine in dimethylformamide (Sigma‐Aldrich, cat. no. 80645‐500ML)
  • Dimethylformamide (DMF; Sigma‐Aldrich, cat. no. 227056‐2L)
  • Dicholoromethane (DCM; Sigma‐Aldrich, cat. no. 270997‐2L)
  • Palmitic acid (Sigma, cat. no. P0500‐10G)
  • Lignoceric acid (Sigma, cat. no. L6641‐1G)
  • N,N‐Diisopropylethylamine (DIPEA) (Sigma, cat. no. 387649‐100ML)
  • N,N′‐Diisopropylcarbodiimide (DIC; Sigma, cat. no. D125407‐25G)
  • Ammonium hydroxide, 28% in water (Sigma, cat. no. 221228‐1 L‐A)
  • Methylamine, 40% in water (Sigma, cat. no. 426466‐1L)
  • 0.1 M triethylammonium acetate (TEAA)
  • Acetonitrile (HPLC‐grade)
  • Polyethylene wash bottles (Sigma, cat. no. Z177024‐6EA)
  • Vacuum concentrator (e.g., SpeedVac system)
  • Empty synthesis columns (Glen Research, cat. no. 20‐0021‐01)
  • Synthesis column frits (Glen Research, cat. no. 20‐0021‐0F)
  • DNA synthesizer (e.g., Expedite 8909 or Biolytic 3900)
  • 1‐liter Pyrex bottles
  • Microcentrifuge tubes with cap locks (e.g., Eppendorf)
  • 0.2 µm Ultrafree‐MC Centrifugal Filter Units (Millipore, cat. no. UFC30GV0S)
  • HPLC system (e.g., Agilent 1200 Series)
  • C8 Column (Hypersil Gold, Thermo Scientific, cat. no. 25205254630)
  • Lyophilizer (e.g., Labconco FreeZone)
  • Spectrophotometer (e.g., Thermo NanoDrop 2000)
  • 1 mmol Universal solid support DNA synthesis resin (Glen Research, cat. no. 20‐5041‐10)
  • 5′‐Amino‐Modifier C6 (Glen Research, cat. no. 10‐1906‐90)

Basic Protocol 4: DNA‐Programmed Assembly of Cells on Patterned Glass

  Materials
  • Patterned slides ( protocol 1)
  • PDMS flow cells (see protocol 9)
  • Priming buffer (see recipe)
  • Flow buffer (see recipe)
  • High‐concentration, DNA‐labeled, ice‐cold cells ( protocol 3)
  • Scotch tape (3 M, “Magic” Tape 810)
  • Microcrystalline wax (Douglas & Sturgess, cat. no. SC‐1159)
  • Mounted toggle clamp (see protocol 10)

Support Protocol 2: Quantification of Cell‐DNA Functionalization

  Additional Materials (also see protocol 3)
  • Phosphate‐buffered saline (e.g., Fisher, cat. no. R58190001A)
  • 5′‐FITC‐DNA with sequence complementary to that on the cells (Eurofins MWG Operon or similar vendor): resuspend in distilled H 2O for a 300 µM stock and store at −20°C in a foil‐wrapped tube
  • LIVE/DEAD Fixable Far Red Dead Cell Stain Kit (Thermo Fisher, cat. no. L10120)
  • Fluorescent microsphere standards, Quantum FITC‐5 MESF (Bangs Laboratories, cat. no. 555)
  • FACS tubes
  • Flow cytometer
  • Additional reagents and equipment for flow cytometry (Robinson et al., )

Support Protocol 3: Measurement of Surface‐DNA Functionalization

  Additional Materials (also see Basic Protocols protocol 11 and protocol 22)
  • 2 mM 20 mer 5′‐amine‐DNA in dH 2O (e.g., available from Eurofins MWG Operon)
  • 20× saline sodium citrate (SSC; Sigma, cat. no. S6639‐1L)
  • 5′‐FITC‐DNA with sequence complementary to that on the slide (Eurofins MWG Operon or similar vendor) (resuspend in dH 2O for a 300 µM stock and store at −20°C)
  • Nexterion AL slide (Applied Microarrays, cat. no. 1064874)
  • Petri dishes
  • Vacuum dessicator
  • 18 × 18–mm coverslips (Fisher Scientific, cat. no. 12‐542A)
  • Fluorescence microscope

Support Protocol 4: Regeneration of DNA‐Patterned Surfaces

  Materials (also see protocol 5)
  • 0.05% (w/v) trypsin (ThermoFisher, cat. no. 25300054)
  • 0.1% (w/v) SDS in distilled H 2O

Support Protocol 5: Preparation of PDMS Flow Cells

  Materials
  • Sylgard 184 (Fisher, cat. no. NC9644388)
  • Diamond scribe (Fisher, cat. no. 17‐467‐634)
  • 18 × 18–mm no. 1 coverslips (Fisher, cat. no. 12‐542A)
  • Permanent double‐sided Scotch tape (3 M, Tape 665)
  • 15‐cm Petri dishes

Support Protocol 6: Construction of Mounted Toggle Clamp

  Materials
  • Sigmacote (Sigma, cat. no. SL2‐25ML)
  • 3/16‐in. acrylic sheet (McMaster‐Carr, cat. no. 8560K163)
  • Laser cutter (e.g., Universal Laser Systems, cat. no. VLS 3.50)
  • Hold‐down toggle clamp (McMaster‐Carr, cat. no. 5126A45)
  • 3/8–in. 8‐32 machine screws (McMaster Carr, cat. no. 91735A192)
  • 36‐grit sandpaper pad (McMaster‐Carr, cat. no. 8221A11)
  • 15‐ml conical tubes (e.g., BD Falcon)
  • 65ºC hot plate or water bath

Basic Protocol 5: 3‐D Transfer into Matrigel

  Materials
  • Matrigel (Corning, cat. no. 356231)
  • Turbo DNase (Life Technologies, cat. no. AM2238)
  • 70% ethanol
  • Cell culture medium
  • Aluminum tube rack (or other sterilizable, chillable, high‐heat‐capacity object; e.g., Labconco, cat. no. 4026402)
  • Toggle clamp assembly with flow cells (from protocol 5)
  • Autoclaved razor blades
  • Lab‐Tek II Chambered Coverglass, 2‐well (Fisher cat.no. 12565336)
  • Self‐closing curved‐tip tweezers (Fisher cat. no. 50‐242‐89)

Alternate Protocol 1: 3‐D Transfer into Agarose

  Additional Materials (also see protocol 11)
  • Agarose Type IX‐A (Sigma, cat. no. A2576)
  • Phosphate‐buffered saline (e.g., Fisher, cat. no. R58190001A)
  • Aluminum tube rack (or other sterilizable, warmable, high‐heat‐capacity object; e.g., Labconco, cat. no. 4026402)
  • 50‐ml flask

Alternate Protocol 2: 3‐D Transfer into Collagen/Matrigel Mixtures

  Additional Materials (also see protocol 11)
  • Sodium hydroxide (Sigma, cat. no, S5881‐500G)
  • Sterile 20× PBS (VWR, cat. no. 101076‐202)
  • High‐concentration rat‐tail collagen I (Corning, cat. no. 354249)
  • pH paper (Fisher, cat. no. 13‐640‐502)
  • 100‐ml beaker, sterile
  • Vacuum desiccator (e.g., 150‐mm Nalgene desiccator, Thermo, cat. no. 24987‐106)
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Figures

Videos

Literature Cited

  Albrecht, D.R., Underhill, G.H., Wassermann, T.B., Sah, R.L., and Bhatia, S.N. 2006. Probing the role of multicellular organization in three‐dimensional microenvironments. Nat. Methods 3:369‐375. doi: 10.1038/nmeth873.
  Bates, T.R., Nightingale, C.H., and Dixon, E. 1973. Kinetics of hydrolysis of polyoxyethylene (20) sorbitan fatty acid ester surfactants. J. Pharm. Pharmacol. 25:470‐477. doi: 10.1111/j.2042‐7158.1973.tb09135.x.
  Cerchiari, A., Garbe, J.C., Todhunter, M.E., Jee, N.Y., Pinney, J.R., LaBarge, M.A., Desai, T.A., and Gartner, Z.J. 2015. Formation of spatially and geometrically controlled three‐dimensional tissues in soft gels by sacrificial micromolding. Tissue Eng. Part C: Methods. 21:541‐547. Available at: http://online.liebertpub.com/doi/abs/10.1089/ten.tec.2014.0450 [Accessed April 8, 2015].
  Chan, H.F., Zhang, Y., Ho, Y.‐P., Chiu, Y.‐L., Jung, Y., and Leong, K.W. 2013. Rapid formation of multicellular spheroids in double‐emulsion droplets with controllable microenvironment. Sci. Rep. Available at: http://www.nature.com/srep/2013/131210/srep03462/full/srep03462.html?message‐global=remove&WT.ec_id=SREP‐631‐20140102 [Accessed July 31, 2015].
  Chen, S., Bremer, A.W., Scheideler, O.J., Na, Y.S., Todhunter, M.E., Hsiao, S., Bomdica, P.R., Maharbiz, M.M., Gartner, Z.J., and Schaffer, D.V. 2016. Interrogating cellular fate decisions with high‐throughput arrays of multiplexed cellular communities. Nat. Commun 7:10309. Available at: http://dx.doi.org/10.1038/ncomms10309.
  Chung, K. and Deisseroth, K. 2013. CLARITY for mapping the nervous system. Nat. Methods 10:508‐513. doi: 10.1038/nmeth.2481.
  Debnath, J., Muthuswamy, S.K., and Brugge, J.S. 2003. Morphogenesis and oncogenesis of MCF‐10A mammary epithelial acini grown in three‐dimensional basement membrane cultures. Methods 30:256‐268. doi: 10.1016/S1046‐2023(03)00032‐X.
  Dhimolea, E., Soto, A.M., and Sonnenschein, C. 2012. Breast epithelial tissue morphology is affected in 3D cultures by species‐specific collagen‐based extracellular matrix. J. Biomed. Mater. Res. Part A 100:2905‐2912. doi: 10.1002/jbm.a.34227.
  El Muslemany, K.M., Twite, A.A., ElSohly, A.M., Obermeyer, A.C., Mathies, R.A., and Francis, M.B. 2014. Photoactivated bioconjugation between ortho‐azidophenols and anilines: A facile approach to biomolecular photopatterning. J. Am. Chem. Soc. 136:12600‐12606. doi: 10.1021/ja503056x.
  Gartner, Z.J. and Bertozzi, C.R. 2009. Programmed assembly of 3‐dimensional microtissues with defined cellular connectivity. Proc. Natl. Acad. Sci. 106:4606‐4610. doi: 10.1073/pnas.0900717106.
  Hsiao, S.C., Shum, B.J., Onoe, H., Douglas, E.S., Gartner, Z.J., Mathies, R.A., Bertozzi, C.R., Francis, M.B. 2009. Direct cell surface modification with DNA for the capture of primary cells and the investigation of myotube formation on defined patterns. Langmuir 25:6985‐6991.
  Hsu, Y.‐H., Moya, M.L., Hughes, C.C.W., George, S.C., and Lee, A.P. 2013. A microfluidic platform for generating large‐scale nearly identical human microphysiological vascularized tissue arrays. Lab on a Chip 13:2990‐2998. doi: 10.1039/c3lc50424g.
  Kinkel, J.N. and Unger, K.K. 1984. Role of solvent and base in the silanization reaction of silicas for reversed‐phase high‐performance liquid chromatography. J. Chromatogr. A. 316:193‐200. doi: 10.1016/S0021‐9673(00)96151‐X.
  Kumar, A., Biebuyck, H.A., and Whitesides, G.M. 1994. Patterning self‐assembled monolayers: Applications in materials science. Langmuir 10:1498‐1511. doi: 10.1021/la00017a030.
  Lange, S.A., Benes, V., Kern, D.P., Hörber, J.K.H., and Bernard, A. 2004. Microcontact printing of DNA molecules. Anal. Chem. 76:1641‐1647. doi: 10.1021/ac035127w.
  Li, C.Y., Stevens, K.R., Schwartz, R.E., Alejandro, B.S., Huang, J.H., and Bhatia, S.N. 2014. Micropatterned cell‐cell interactions enable functional encapsulation of primary hepatocytes in hydrogel microtissues. Tissue Eng. Part A 20:2200‐2212. doi: 10.1089/ten.tea.2013.0667.
  Markham, N.R. and Zuker, M. 2005. DINAMelt web server for nucleic acid melting prediction. Nucleic Acids Res. 33:W577‐W581. doi: 10.1093/nar/gki591.
  Matsusaki, M., Sakaue, K., Kadowaki, K., and Akashi, M. 2013. Three‐dimensional human tissue chips fabricated by rapid and automatic inkjet cell printing. Adv. Healthc. Mater. 2:534‐539. doi: 10.1002/adhm.201200299.
  Monserud, J.H. and Schwartz, D.K. 2012. Effects of molecular size and surface hydrophobicity on oligonucleotide interfacial dynamics. Biomacromolecules 13:4002‐4011. doi: 10.1021/bm301289n.
  Murphy, S.V. and Atala, A. 2014. 3D bioprinting of tissues and organs. Nat. Biotechnol. 32:773‐785. doi: 10.1038/nbt.2958.
  Nelson, C.M., VanDuijn, M.M., Inman, J.L., Fletcher, D.A., and Bissell, M.J. 2006. Tissue geometry determines sites of mammary branching morphogenesis in organotypic cultures. Science 314:298‐300. doi: 10.1126/science.1131000.
  Onoe, H., Hsiao, S.C., Douglas, E.S., Gartner, Z.J., Bertozzi, C.R., Francis, M.B., and Mathies, R.A. 2012. Cellular microfabrication: Observing intercellular interactions using lithographically‐defined DNA capture sequences. Langmuir 28:8120‐8126. doi: 10.1021/la204863s.
  Phelan, K. and May, K.M. 2015. Basic techniques in mammalian cell tissue culture. Curr. Protoc. Cell Biol. 66:1.1.1‐1.1.22. doi: 10.1002/0471143030.cb0101s66.
  Robinson, J.P., Darzynkiewicz, Z., Hoffman, R., Nolan, J.P., Orfao, A., Rabinovitch, P.S., and Watkins, S. (eds.) 2016. Current Protocols in Cytometry. John Wiley & Sons, Hoboken, N.J.
  Selden, N.S., Todhunter, M.E., Jee, N.Y., Liu, J.S., Broaders, K.E., and Gartner, Z.J. 2012. Chemically programmed cell adhesion with membrane‐anchored oligonucleotides. J. Am. Chem. Soc. 134:765‐768. doi: 10.1021/ja2080949.
  Stevens, K.R., Ungrin, M.D., Schwartz, R.E., Ng, S., Carvalho, B., Christine, K.S., Chaturvedi, R.R., Li, C.Y., Zandstra, P.W., Chen, C.S., and Bhatia, S.N. 2013. InVERT molding for scalable control of tissue microarchitecture. Nat. Commun. 4:1847. doi: 10.1038/ncomms2853.
  Twite, A.A., Hsiao, S.C., Onoe, H., Mathies, R.A., and Francis, M.B. 2012. Direct attachment of microbial organisms to material surfaces through sequence‐specific DNA hybridization. Adv. Mater. 24:2380‐2385. doi: 10.1002/adma.201104336.
  Weber, R.J., Liang, S.I., Selden, N.S., Desai, T.A., and Gartner, Z.J. 2014. Efficient targeting of fatty‐acid modified oligonucleotides to live cell membranes through stepwise assembly. Biomacromolecules 15:4621‐4626. doi: 10.1021/bm501467h. Epub 2014 Nov 20.
  Whipple, R.A., Cheung, A.M., and Martin, S.S. 2007. Detyrosinated microtubule protrusions in suspended mammary epithelial cells promote reattachment. Exp. Cell Res. 313:1326‐1336. doi: 10.1016/j.yexcr.2007.02.001.
Key References
  Todhunter, M.E., Jee, N.Y., Hughes, A.J., Coyle, M.C., Cerchiari, A., Farlow, J., Garbe, J.C., LaBarge, M.A., Desai, T.A., and Gartner, Z.J. 2015. Programmed synthesis of 3D tissues. Nat. Methods 12:975‐981. doi: 10.1038/nmeth.3553.
  This reference details a variety of DPAC‐made organoids, describing both their constituent cells and synthetic schemes.
Internet Resources
  http://bioforcenano.com/resources/aapplication‐note‐203‐speed‐printing‐or‐printing‐in‐no‐laser‐mode‐with‐the‐nano‐enabler‐system/
  Application Note 203: “Speed Printing”; or, printing in “No Laser Mode” with the Nano eNabler System BioForce Nanosciences.
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